Methods and Compositions for Improved F-18 Labeling of Proteins, Peptides and Other Molecules

ABSTRACT

The present application discloses compositions and methods of synthesis and use of  18 F- or  19 F-labeled molecules of use in PET, SPECT and/or MR imaging. Preferably, the  18 F or  19 F is conjugated to a targeting molecule by formation of a complex with a group IIIA metal and binding of the complex to a bifunctional chelating agent, which may then be directly or indirectly attached to the targeting molecule. In other embodiments, the  18 F or  19 F labeled moiety may comprise a targetable construct used in combination with a bispecific antibody to target a disease-associated antigen. The disclosed methods and compositions allow the simple and reproducible labeling of molecules at very high efficiency and specific activity in 30 minutes or less. In preferred embodiments, the bifunctional chelating agent bound to  18 F- or  19 F-metal complex may be conjugated to the molecule to be labeled at a reduced temperature, e.g. room temperature.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/897,849, filed May 20, 2013, which was a continuation-in-part of U.S.patent application Ser. No. 13/850,591 (now U.S. Pat. No. 8,617,518),filed Mar. 26, 2013, which was a divisional of Ser. No. 13/474,260 (nowU.S. Pat. No. 8,444,956), filed May 17, 2012, which was a divisional ofSer. No. 12/958,889 (now U.S. Pat. No. 8,202,509), filed Dec. 2, 2010,which was a continuation-in-part of Ser. No. 12/433,212 (now U.S. Pat.No. 8,153,100), filed Apr. 30, 2009, which was a continuation-in-part ofSer. No. 12/343,655 (now U.S. Pat. No. 7,993,626), filed Dec. 24, 2008,which was a continuation-in-part of Ser. No. 12/112,289 (now U.S. Pat.No. 7,563,433), filed Apr. 30, 2008, which was a continuation-in-part ofSer. No. 11/960,262 (now U.S. Pat. No. 7,597,876), filed Dec. 19, 2007,which claimed the benefit under 35 U.S.C. 119(e) of Provisional U.S.Patent Application No. 60/884,521, filed Jan. 11, 2007. U.S. Ser. No.13/897,849 is continuation-in-part of U.S. patent application Ser. No.13/752,877 (now U.S. Pat. No. 8,496,912), filed Jan. 29, 2013, which wasa divisional of Ser. No. 13/309,714 (now U.S. Pat. No. 8,398,956), filedDec. 2, 2011, which was a continuation-in-part of Ser. No. 12/958,889(now U.S. Pat. No. 8,202,509), filed Dec. 2, 2010, which was acontinuation-in-part of Ser. No. 12/433,212 (now U.S. Pat. No.8,153,100), filed Apr. 30, 2009, which was a continuation-in-part ofSer. No. 12/343,655 (now U.S. Pat. No. 7,993,626), filed Dec. 24, 2008,which was a continuation-in-part of Ser. No. 12/112,289 (now U.S. Pat.No. 7,563,433), filed Apr. 30, 2008, which was a continuation-in-part ofSer. No. 11/960,262 (now U.S. Pat. No. 7,597,876), filed Dec. 19, 2007,which claimed the benefit under 35 U.S.C. 119(e) of Provisional U.S.Patent Application No. 60/884,521, filed Jan. 11, 2007. U.S. Ser. No.13/897,849 is a continuation-in-part of U.S. patent application Ser. No.13/323,139 (now U.S. Pat. No. 8,545,809), filed Dec. 12, 2011, whichclaimed the benefit under 35 U.S.C. 119(e) of Provisional U.S. PatentApplication Nos. 61/422,258, filed Dec. 13, 2010; 61/479,660, filed Apr.27, 2011; 61/492,613, filed Jun. 2, 2011; 61/523,668, filed Aug. 15,2011; 61/540,248, filed Sep. 28, 2011; 61/547,434, filed Oct. 14, 2011,and also was a continuation-in-part of Ser. No. 13/309,714 (now U.S.Pat. No. 8,398,956), filed Dec. 2, 2011, which claimed the benefit under35 U.S.C. 119(e) of Provisional U.S. Patent Application No. 61/419,082,filed Dec. 2, 2010. Ser. No. 13/309,714 was a continuation-in-part ofSer. No. 12/958,889 (now U.S. Pat. No. 8,202,509), filed Dec. 2, 2010,which claimed the benefit under 35 U.S.C. 119(e) of Provisional U.S.Patent Application Nos. 61/266,773, filed Dec. 4, 2009; 61/302,280,filed Feb. 8, 2010; 61/316,125, filed Mar. 22, 2010; 61/347,486, filedMay 24, 2010; 61/381,720, filed Sep. 10, 2010; 61/388,268, filed Sep.30, 2010. U.S. Ser. No. 12/958,889 was also a continuation-in-part ofSer. No. 12/433,212 (now issued U.S. Pat. No. 8,153,100), filed Apr. 30,2009, which was a continuation-in-part of Ser. No. 12/343,655 (nowissued U.S. Pat. No. 7,993,626), filed Dec. 24, 2008, which was acontinuation-in-part of Ser. No. 12/112,289 (now issued U.S. Pat. No.7,563,433), filed Apr. 30, 2008, which was a continuation-in-part ofSer. No. 11/960,262 (now issued U.S. Pat. No. 7,597,876), filed Dec. 19,2007, which claimed the benefit under 35 U.S.C. 119(e) of ProvisionalU.S. Patent Application No. 60/884,521, filed Jan. 11, 2007. U.S. Ser.No. 13/897,849 claims the benefit under 35 U.S.C. 119(e) of ProvisionalU.S. Patent Application No. 61/649,526, filed May 21, 2012. The text ofeach priority application is incorporated herein by reference in itsentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 20, 2013, isnamed IMM310US15_SL.txt and is 28,825 bytes in size.

FIELD

The present invention concerns methods of labeling peptides or othermolecules with ¹⁸F or ¹⁹F that are of use, for example, in PET or NMR invivo imaging. Preferably, the ¹⁸F or ¹⁹F is attached as a complex withaluminum or another metal, such as a Group IIIA metal, via a chelatingmoiety, which may be covalently linked to a protein, peptide or othermolecule. The chelating moiety may be attached to a protein, peptide orother molecule either before or after binding to the metal-¹⁸F ormetal-¹⁹F complex. Although labeling may occur at an elevatedtemperature, such as 70° C., 80° C., 90° C., 95° C., 100° C., 105° C.,110° C., or any temperature in between, preferably labeling of heatsensitive molecules may occur at a lower temperature, such as roomtemperature.

In certain embodiments, the labeled molecule may be used for targeting acell, tissue, organ or pathogen to be imaged or detected. Exemplarytargeting molecules include, but are not limited to, an antibody,antigen-binding antibody fragment, bispecific antibody, affibody,diabody, minibody, ScFvs, aptamer, avimer, targeting peptide,somatostatin, bombesin, octreotide, RGD peptide, folate, folate analogor any other molecule known to bind to a disease-associated target.

Using the techniques described herein, ¹⁸F-labeled molecules of highspecific activity may be prepared in 30 minutes or less and are suitablefor use in imaging techniques without the need for HPLC purification ofthe labeled molecule. Labeling may occur in a saline medium suitable fordirect use in vivo. In alternative embodiments an organic solvent may beadded to improve the labeling efficiency. The labeled molecules arestable under physiological conditions, although for certain purposes,such as kit formulations, a stabilizing agent such as ascorbic acid,trehalose, sorbitol or mannitol may be added. In other alternativeembodiments, a chelating moiety may be preloaded with aluminum andlyophilized for storage, prior to labeling with ¹⁸F.

BACKGROUND

Positron Emission Tomography (PET) has become one of the most prominentfunctional imaging modalities in diagnostic medicine, with very highsensitivity (fmol), high resolution (4-10 mm) and tissue accretion thatcan be adequately quantitated (Volkow et al., 1988, Am. J. Physiol.Imaging 3:142). Although [¹⁸F]2-deoxy-2-fluoro-D-glucose ([¹⁸F]FDG) isthe most widely used PET imaging agent in oncology (Fletcher et al.,2008, J. Nucl. Med. 49:480), there is a keen interest in developingother labeled compounds for functional imaging to complement and augmentanatomic imaging methods (Torigian et al., 2007, CA Cancer J. Clin.57:206), especially with the hybrid PET/computed tomography systemscurrently in use. Thus, there is a need to have facile methods ofconjugating positron emitting radionuclides to various molecules ofbiological and medical interest.

Peptides or other targeting molecules can be labeled with the positronemitters ¹⁸F, ⁶⁴Cu, ¹¹C, ⁶⁶Ga, ⁶⁸Ga, ⁷⁶Br, ^(94m)Tc, ⁸⁶Y, and ¹²⁴I. Alow ejection energy for a PET isotope is desirable to minimize thedistance that the positron travels from the target site before itgenerates the two 511 keV gamma rays that are imaged by the PET camera.Many isotopes that emit positrons also have other emissions such asgamma rays, alpha particles or beta particles in their decay chain. Itis desirable to have a PET isotope that is a pure positron emitter sothat any dosimetry problems will be minimized. The half-life of theisotope is also important, since the half-life must be long enough toattach the isotope to a targeting molecule, analyze the product, injectit into the patient, and allow the product to localize, clear fromnon-target tissues and then image. ¹⁸F (β⁺97%, 635 keV, t_(1/2) 110 min)is one of the most widely used PET emitting isotopes because of its lowpositron emission energy, lack of side emissions and suitable half-life.

Conventionally, ¹⁸F is attached to compounds by binding it to a carbonatom (Miller et al., 2008, Angew Chem Int Ed 47:8998-9033), butattachments to silicon (Shirrmacher et al., 2007, Bioconj Chem18:2085-89; Hohne et al., 2008, Bioconj Chem 19:1871-79) and boron (Tinget al., 2008, Fluorine Chem 129:349-58) have also been reported. Bindingto carbon usually involves multistep syntheses, including multiplepurification steps, which is problematic for an isotope with a 110-minhalf-life, and typically results in poor radiochemical yields. Currentmethods for ¹⁸F-labeling of peptides typically involve the labeling of areagent at low specific activity, HPLC purification of the reagent andthen conjugation to the peptide of interest. The conjugate is oftenrepurified after conjugation to obtain the desired specific activity oflabeled peptide.

An example is the labeling method of Poethko et al. (J. Nucl. Med. 2004;45: 892-902) in which 4-[¹⁸F]fluorobenzaldehyde is first synthesized andpurified (Wilson et al, J. Labeled Compounds and Radiopharm. 1990;XXVIII: 1189-1199) and then conjugated to the peptide. The peptideconjugate is then purified by HPLC to remove excess peptide that wasused to drive the conjugation to completion. Other examples includelabeling with succinimidyl [¹⁸F]fluorobenzoate (SFB) (e.g., Vaidyanathanet al., 1992, Int. J. Rad. Appl. Instrum. B 19:275), other acylcompounds (Tada et al., 1989, Labeled Compd. Radiopharm.XXVII:1317;Wester et al., 1996, Nucl. Med. Biol. 23:365; Guhlke et al., 1994, Nucl.Med. Biol 21:819), or click chemistry adducts (Li et al., 2007, BioconjChem. 18:1987). The total synthesis and formulation time for thesemethods ranges between 1-3 hours, with most of the time dedicated to theHPLC purification of the labeled peptides to obtain the specificactivity required for in vivo targeting. With a 2 hr half-life, all ofthe manipulations that are needed to attach the ¹⁸F to the peptide are asignificant burden. These methods are also tedious to perform andrequire the use of equipment designed specifically to produce thelabeled product and/or the efforts of specialized professional chemists.They are also not conducive to kit formulations that could routinely beused in a clinical setting.

A need exists for a rapid, simple method of ¹⁸F labeling of targetingmoieties, such as proteins or peptides, preferably at high radiochemicalyield, which results in targeting constructs of suitable specificactivity and in vivo stability for detection and/or imaging, whileminimizing the requirements for specialized equipment or highly trainedpersonnel and reducing operator exposure to high levels of radiation.More preferably a need exists for methods of preparing ¹⁸F-labeledtargeting peptides of use in pretargeting technologies. A further needexists for prepackaged kits that could provide compositions required forperforming such novel methods. An additional need exists for methods ofefficiently labeling temperature sensitive molecules.

SUMMARY

In various embodiments, the present invention concerns compositions andmethods relating to ¹⁸F- or ¹⁹F-labeled molecules of use for PET or NMRimaging. As discussed herein, where the present application refers to¹⁸F the skilled artisan will realize that either ¹⁸F, ¹⁹F or anotherradionuclide, such as ⁶⁸Ga, may be utilized. In an exemplary approach,the ¹⁸F is bound to a metal and the ¹⁸F-metal complex is attached to aligand on a peptide or other molecule. As described below, the metals ofgroup IIIA (aluminum, gallium, indium, and thallium) are suitable for¹⁸F binding, although aluminum is preferred. Lutetium may also be ofuse. The metal binding ligand is preferably a chelating agent, such asNOTA, NODA, NETA, DOTA, DTPA and other chelating groups discussed inmore detail below. Alternatively, one can attach the metal to a moleculefirst and then add the ¹⁸F to bind to the metal. In still otherembodiments, one may attach an ¹⁸F-metal to a chelating moiety first andthen attach the labeled chelating moiety to a molecule, such as atemperature sensitive molecule. In this way, the ¹⁸F-metal may beattached to a chelating moiety at a higher temperature, such as between90° to 110° C., more preferably between 95° to 105° C., and the¹⁸F-labeled chelating moiety may be attached to a temperature sensitivemolecule at a lower temperature, such as at room temperature. Inpreferred embodiments, the labeling method uses a biofunctional chelatorthat forms a physiologically stable complex with metal-¹⁸F, whichcontains reactive groups that can bind to proteins, peptides or othertargeting molecules at, e.g., room temperature. More preferably,labeling can be accomplished in 10 to 15 minutes in aqueous medium, witha total synthesis time of about 30 minutes.

The skilled artisan will realize that virtually any delivery moleculecan be attached to ¹⁸F for imaging purposes, so long as it containsderivatizable groups that may be modified without affecting theligand-receptor binding interaction between the delivery molecule andthe cellular or tissue target receptor. Although the Examples belowprimarily concern ¹⁸F-labeled peptide moieties, many other types ofdelivery molecules, such as oligonucleotides, hormones, growth factors,cytokines, chemokines, angiogenic factors, anti-angiogenic factors,immunomodulators, proteins, nucleic acids, antibodies, antibodyfragments, drugs, interleukins, interferons, oligosaccharides,polysaccharides, siderophores, lipids, etc. may be ¹⁸F-labeled andutilized for imaging purposes.

Exemplary targetable construct peptides described in the Examples below,of use for pre-targeting delivery of ¹⁸F or other agents, include butare not limited to IMP449, IMP460, IMP461, IMP467, IMP469, IMP470,IMP471, IMP479, IMP485, IMP486, IMP487, IMP488, IMP490, IMP493, IMP495,IMP497, IMP500, IMP508, IMP517, comprising chelating moieties thatinclude, but are not limited to, DTPA, NOTA, benzyl-NOTA, alkyl or arylderivatives of NOTA, NODA, NODA-GA, C-NETA, succinyl-C-NETA andbis-t-butyl-NODA. In a preferred embodiment, a chelating moiety based onNODA-propyl amine (e.g., (tBu)₂NODA-propyl amine) may be derivatized toform a reactive thiol, maleimide, azide, alkyne or aminooxy group, whichmay then be conjugated to a targeting molecule at a reduced temperaturevia azide-alkyne coupling, thioether, amide, dithiocarbamate,thiocarbamate, oxime or thiourea formation.

In certain embodiments, the exemplary ¹⁸F-labeled peptides may be of useas targetable constructs in a pre-targeting method, utilizing bispecificor multispecific antibodies or antibody fragments. In this case, theantibody or fragment will comprise one or more binding sites for atarget associated with a disease or condition, such as atumor-associated or autoimmune disease-associated antigen or an antigenproduced or displayed by a pathogenic organism, such as a virus,bacterium, fungus or other microorganism. A second binding site willspecifically bind to the targetable construct. Methods for pre-targetingusing bispecific or multispecific antibodies are well known in the art(see, e.g., U.S. Pat. No. 6,962,702, the Examples section of which isincorporated herein by reference.) Similarly, antibodies or fragmentsthereof that bind to targetable constructs are also well known in theart, such as the 679 monoclonal antibody that binds to HSG (histaminesuccinyl glycine) or the 734 antibody that binds to In-DTPA (see U.S.Pat. Nos. 7,429,381; 7,563,439; 7,666,415; and 7,534,431, the Examplessection of each incorporated herein by reference). Generally, inpretargeting methods the bispecific or multispecific antibody isadministered first and allowed to bind to cell or tissue targetantigens. After an appropriate amount of time for unbound antibody toclear from circulation, the e.g. ¹⁸F-labeled targetable construct isadministered to the patient and binds to the antibody localized totarget cells or tissues. Then an image is taken, for example by PETscanning.

In alternative embodiments, molecules that bind directly to receptors,such as somatostatin, octreotide, bombesin, folate or a folate analog,an RGD peptide or other known receptor ligands may be labeled and usedfor imaging. Receptor targeting agents may include, for example, TA138,a non-peptide antagonist for the integrin α_(v)β₃ receptor (Liu et al.,2003, Bioconj. Chem. 14:1052-56). Other methods of receptor targetingimaging using metal chelates are known in the art and may be utilized inthe practice of the claimed methods (see, e.g., Andre et al., 2002, J.Inorg. Biochem. 88:1-6; Pearson et al., 1996, J. Med. Chem. 39:1361-71).

The type of diseases or conditions that may be imaged is limited only bythe availability of a suitable delivery molecule for targeting a cell ortissue associated with the disease or condition. Many such deliverymolecules are known. For example, any protein or peptide that binds to adiseased tissue or target, such as cancer, may be labeled with ¹⁸F bythe disclosed methods and used for detection and/or imaging. In certainembodiments, such proteins or peptides may include, but are not limitedto, antibodies or antibody fragments that bind to tumor-associatedantigens (TAAs). Any known TAA-binding antibody or fragment may belabeled with ¹⁸F by the described methods and used for imaging and/ordetection of tumors, for example by PET scanning or other knowntechniques.

Certain alternative embodiments involve the use of “click” chemistry forattachment of ¹⁸F-labeled moieties to targeting molecules. Preferably,the click chemistry involves the reaction of a targeting molecule suchas an antibody or antigen-binding antibody fragment, comprising afunctional group such as an alkyne, nitrone or an azide group, with a¹⁸F-labeled moiety comprising the corresponding reactive moiety such asan azide, alkyne or nitrone. Where the targeting molecule comprises analkyne, the chelating moiety or carrier will comprise an azide, anitrone or similar reactive moiety. The click chemistry reaction mayoccur in vitro to form a highly stable, ¹⁸F-labeled targeting moleculethat is then administered to a subject.

In other alternative embodiments, a prosthetic group, such as aNODA-maleimide moiety, may be labeled with ¹⁸F-metal and then conjugatedto a targeting molecule, for example by a maleimide-sulfhydryl reaction.Exemplary NODA-maleimide moieties include, but are not limited to,NODA-MPAEM, NODA-PM, NODA-PAEM, NODA-BAEM, NODA-BM, NODA-MPM, andNODA-MBEM.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Figures are included to illustrate particular embodimentsof the invention and are not meant to be limiting as to the scope of theclaimed subject matter.

FIG. 1. Biodistribution of ¹⁸F-labeled agents in tumor-bearing nude miceby microPET imaging. Coronal slices of 3 nude mice bearing a small,subcutaneous LS174T tumor on each left flank after being injected witheither (A) ¹⁸F-FDG, (B) Al¹⁸F(IMP449) pretargeted with theanti-CEA×anti-HSG bsMAb, (C) Al¹⁸F(IMP449) alone (not pretargeted withthe bsMAb). Biodistribution data expressed as percent-injected dose pergram (% ID/g) are given for the tissues removed from the animals at theconclusion of the imaging session. Abbreviations: B, bone marrow; H,heart; K, kidney; T, tumor.

FIG. 2. Dynamic imaging study of pretargeted Al¹⁸F(IMP449) given to anude mouse bearing a 35-mg LS174T human colorectal cancer xenograft inthe upper flank. The top 3 panels show coronal, sagittal, and transversesections, respectively, taken of a region of the body centering on thetumor's peripheral location at 6 different 5-min intervals over the120-min imaging session. The first image on the left in each sectionalview shows the positioning of the tumor at the intersection of thecrosshairs, which is highlighted by arrows. The animal was partiallytilted to its right side during the imaging session. The bottom 2 panelsshow additional coronal and sagittal sections that focus on a moreanterior plane in the coronal section to highlight distribution in theliver and intestines, while the sagittal view crosses more centrally inthe body. Abbreviations: Cor, coronal; FA, forearms; H, heart; K,kidney; Lv, liver; Sag, sagittal; Tr, transverse; UB, urinary bladder.

FIG. 3. In vivo tissue distribution with Al¹⁸F(IMP466) bombesinanalogue.

FIG. 4. Comparison of biodistribution of Al¹⁸F(IMP466) and ⁶⁸Ga(IMP466)at 2 hours post-injection in AR42J tumor-bearing mice (n=5). As acontrol, mice in separate groups (n=5) received an excess of unlabeledoctreotide to demonstrate receptor specificity.

FIG. 5. Coronal slices of PET/CT scan of Al¹⁸F(IMP466) and ⁶⁸Ga(IMP466)at 2 hours post-injection in mice with an s.c. AR42J tumor in the neck.Accumulation in tumor and kidneys is clearly visualized.

FIG. 6. Biodistribution of 6.0 nmol ¹²⁵I-TF2 (0.37 MBq) and 0.25 nmol⁶⁸Ga(IMP288) (5 MBq), 1 hour after i.v. injection of ⁶⁸Ga(IMP288) inBALB/c nude mice with a subcutaneous LS174T and SK-RC52 tumor. Valuesare given as means±standard deviation (n=5).

FIG. 7. Biodistribution of 5 MBq FDG and of 5 MBq ⁶⁸Ga(IMP288) (0.25nmol) 1 hour after i.v. injection following pretargeting with 6.0 nmolTF2. Values are given as means±standard deviation (n=5).

FIG. 8. PET/CT images of a BALB/c nude mouse with a subcutaneous LS 174Ttumor (0.1 g) on the right hind leg (light arrow) and a inflammation inthe left thigh muscle (dark arrow), that received 5 MBq ¹⁸F-FDG, and oneday later 6.0 nmol TF2 and 5 MBq ⁶⁸Ga(IMP288) (0.25 nmol) with a 16 hourinterval. The animal was imaged one hour after the ¹⁸F-FDG and⁶⁸Ga(IMP288) injection. The panel shows the 3D volume rendering (A),transverse sections of the tumor region (B) of the FDG-PET scan, and the3D volume rendering (C), transverse sections of the tumor region (D) ofthe pretargeted immunoPET scan.

FIG. 9. Biodistribution of 0.25 nmol Al¹⁸F(IMP449) (5 MBq) 1 hour afteri.v. injection, following 6.0 nmol TF2 administered 16 hours earlier.Biodistribution of Al¹⁸F(IMP449) without pretargeting, orbiodistribution of [Al¹⁸F]. Values are given as means±standarddeviation.

FIG. 10. Static PET/CT imaging study of a BALB/c nude mouse with asubcutaneous LS174T tumor (0.1 g) on the right side (arrow), thatreceived 6.0 nmol TF2 and 0.25 nmol Al¹⁸F(IMP449) (5 MBq) intravenouslywith a 16 hour interval. The animal was imaged one hour after injectionof Al¹⁸F(IMP449). The panel shows the 3D volume rendering (A) posteriorview, and cross sections at the tumor region, (B) coronal, (C) sagittal.

FIG. 11. Structure of IMP479 (SEQ ID NO:54).

FIG. 12. Structure of IMP485 (SEQ ID NO:55).

FIG. 13. Structures of (A) IMP487 (SEQ ID NO:56); (B) IMP490 (SEQ IDNO:52); (C) IMP493 (SEQ ID NO:53); (D) IMP495 (SEQ ID NO: 57); (E)IMP496 (SEQ ID NO: 58); and (F) IMP500.

FIG. 14. Synthesis of bis-t-butyl-NODA-MPAA.

FIG. 15. Synthesis of maleimide conjugate of NOTA.

FIG. 16. Chemical structure of exemplary NODA-based bifunctionalchelators.

FIG. 17. Chemical structures of NODA-BM derived bifunctional chelators.

FIG. 18. Further exemplary structures of NODA-based bifunctionalchelators: (A) NODA-HA, (B) NODA-MPN, (C) NODA-EPN, (D) NODA-MBA, (E)NODA-EPA, (F) NODA-MPAA, (G) NODA-BAEM, (H) NODA-MPAEM, (I) NODA-BM, (J)NODA-MBEM, (K) NODA moiety with maleimide reactive group, (L)alternative NODA moiety with maleimide reactive group, (M) NODA-BA, (N)NODA-EA, (0) NODA-MPH, (P) NODA-butyne, (Q) NODA-MPAPEG₃N₃, (R) NODAmoiety with carboxyl reactive group, (S) NODA moiety with nitrophenylreactive group, (T) NODA moiety with carboxyl and nitrophenyl reactivegroups, (U) another NODA moiety with carboxyl reactive group, (V)another NODA moiety with carboxyl reactive group, (W) another NODAmoiety with carboxyl reactive group, (X) another NODA moiety withcarboxyl reactive group, (Y) another NODA moiety with carboxyl reactivegroup, (Z) another NODA moiety with carboxyl reactive group, (AA)another NODA moiety with carboxyl reactive group, (BB) another NODAmoiety with carboxyl reactive group, (CC) another NODA moiety withcarboxyl reactive group.

FIG. 19(A) and 19(B). Radiochromatograms of the ¹⁸F-labeledfunctionalized TACN ligands.

FIG. 20(A) and 20(B). Radiochromatograms of ¹⁸F-hMN14-Fab′, itsstability in human serum and immunoreactivity with CEA.

FIG. 21. Schematic diagram of automated synthesis module for¹⁸F-labeling via [Al¹⁸F]-chelation.

FIG. 22. NODA-propyl amine derived bifunctional chelating moieties.

FIG. 23. (A) Structure of IMP 508 (SEQ ID NO: 59). (B) Structure ofIMP517 (SEQ ID NO: 60). (C) NODA-2-nitroimidazole. (D)NOTA-DUPA-Peptide.

FIG. 24. Labeling efficiency as a function of temperature.

DETAILED DESCRIPTION

The following definitions are provided to facilitate understanding ofthe disclosure herein. Terms that are not explicitly defined are usedaccording to their plain and ordinary meaning.

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein, the terms “and” and “or” may be used to mean either theconjunctive or disjunctive. That is, both terms should be understood asequivalent to “and/or” unless otherwise stated.

As used herein, “about” means within plus or minus ten percent of anumber. For example, “about 100” would refer to any number between 90and 110.

As used herein, a “peptide” refers to any sequence of naturallyoccurring or non-naturally occurring amino acids of between 2 and 100amino acid residues in length, more preferably between 2 and 10, morepreferably between 2 and 6 amino acids in length. An “amino acid” may bean L-amino acid, a D-amino acid, an amino acid analogue, an amino acidderivative or an amino acid mimetic.

As used herein, the term “pathogen” includes, but is not limited tofungi, viruses, parasites and bacteria, including but not limited tohuman immunodeficiency virus (HIV), herpes virus, cytomegalovirus,rabies virus, influenza virus, hepatitis B virus, Sendai virus, felineleukemia virus, Reovirus, polio virus, human serum parvo-like virus,simian virus 40, respiratory syncytial virus, mouse mammary tumor virus,Varicella-Zoster virus, Dengue virus, rubella virus, measles virus,adenovirus, human T-cell leukemia viruses, Epstein-Barr virus, murineleukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus,lymphocytic choriomeningitis virus, wart virus, blue tongue virus,Streptococcus agalactiae, Legionella pneumophila, Streptococcuspyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseriameningitidis, Pneumococcus, Hemophilus influenzae B, Treponema pallidum,Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae,Brucella abortus, Mycobacterium tuberculosis and Clostridium tetani.

As used herein, a “radiolysis protection agent” refers to any molecule,compound or composition that may be added to an ¹⁸F-labeled complex ormolecule to decrease the rate of breakdown of the ¹⁸F-labeled complex ormolecule by radiolysis. Any known radiolysis protection agent, includingbut not limited to ascorbic acid, may be used.

¹⁸F Labeling Techniques

A variety of techniques for labeling molecules with ¹⁸F are known. Table1 lists the properties of several of the more commonly reportedfluorination procedures. Peptide labeling through carbon often involves¹⁸F-binding to a prosthetic group through nucleophilic substitution,usually in 2- or 3-steps where the prosthetic group is labeled andpurified, attached to the compound, and then purified again. Thisgeneral method has been used to attach prosthetic groups through amidebonds, aldehydes, and “click” chemistry (Marik et al., 2006, BioconjChem 17:1017-21; Poethko et al., 2004, J Nucl Med 45:892-902; Li et al.,2007, Bioconj Chem 18:989-93). The most common amide bond-formingreagent has been N-succinimidyl 4-¹⁸F-fluorobenzoate (¹⁸F-SFB), but anumber of other groups have been tested (Marik et al., 2006). In somecases, such as when ¹⁸F-labeled active ester amide-forming groups areused, it may be necessary to protect certain groups on a peptide duringthe coupling reaction, after which they are cleaved. The synthesis ofthis ¹⁸F-SFB reagent and subsequent conjugation to the peptide requiresmany synthetic steps and takes about 2-3 h.

A simpler, more efficient ¹⁸F-peptide labeling method was developed byPoethko et al. (2004), where a 4-¹⁸F-fluorobenzaldehyde reagent wasconjugated to a peptide through an oxime linkage in about 75-90 min,including the dry-down step. The newer “click chemistry” method attaches¹⁸F-labeled molecules onto peptides with an acetylene or azide in thepresence of a copper catalyst (Li et al, 2007; Glaser and Arstad, 2007,Bioconj Chem 18:989-93). The reaction between the azide and acetylenegroups forms a triazole connection, which is quite stable and forms veryefficiently on peptides without the need for protecting groups. Clickchemistry produces the ¹⁸F-labeled peptides in good yield (˜50%) inabout 75-90 min with the dry-down step.

TABLE 1 Summary of selected ¹⁸F-labeling methods. Höhne et Glaser &Schirrmacher al. Li et al. Arstad Poethko et al. Marik et Author/Ref. etal. (2007) (2008) (2007) (2007) (2004) al (2006) Attachment SiliconSilicon Click Click Aldehyde/oxime Amide Rx steps 2 1 2 2 2 many Rx time40 115-155 110 65-80 75-90 min 110⁺ (min)^(a) (estimated) (estimated)Yield^(b) 55% 13% 54% 50% 40% 10% HPLC- 1 1 2 1 + 1 2 purificationdistillation steps Specific 225-680 62 high high high high Activity(GBq/μmol) ^(a)Including dry-down time ^(b)Decay corrected

A more recent method of binding ¹⁸F to silicon uses isotopic exchange todisplace ¹⁹F with ¹⁸F (Shirrmacher et al., 2007). Performed at roomtemperature in 10 min, this reaction produces the ¹⁸F-prostheticaldehyde group with high specific activity (225-680 GBq/μmol;6,100-18,400 Ci/mmol). The ¹⁸F-labeled aldehyde is subsequentlyconjugated to a peptide and purified by HPLC, and the purified labeledpeptide is obtained within 40 min (including dry-down) with ˜55% yield.This was modified subsequently to a single-step process by incorporatingthe silicon into the peptide before the labeling reaction (Hohne et al,2008). However, biodistribution studies in mice with an¹⁸F-silicon-bombesin derivative showed bone uptake increasing over time(1.35±0.47% injected dose (ID)/g at 0.5 h vs. 5.14±2.71% ID/g at 4.0 h),suggesting a release of ¹⁸F from the peptide, since unbound ¹⁸F is knownto localize in bone (Hohne et al., 2008). HPLC analysis of urine showeda substantial amount of ¹⁸F activity in the void volume, whichpresumably is due to fluoride anion (¹⁸F⁻) released from the peptide. Itwould therefore appear that the ¹⁸F-silicon labeled molecule was notstable in serum. Substantial hepatobiliary excretion was also reported,attributed to the lipophilic nature of the ¹⁸F-silicon-bindingsubstrate, and requiring future derivatives to be more hydrophilic.Methods of attaching ¹⁸F to boron also have been explored; however, thecurrent process produces conjugates with low specific activity (Ting etal., 2008).

Antibodies and peptides are coupled routinely with radiometals,typically in 15 min and in quantitative yields (Meares et al., 1984, AccChem Res 17:202-209; Scheinberg et al., 1982, Science 215:1511-13). ForPET imaging, ⁶⁴Cu and ⁶⁸Ga have been bound to peptides via a chelate,and have shown reasonably good PET-imaging properties (Heppler et al.,2000, Current Med Chem 7:971-94). Since fluoride binds to most metals,we sought to determine if an ¹⁸F-metal complex could be bound to achelator on a targeting molecule (Tewson, 1989, Nucl Med Biol.16:533-51; Martin, 1996, Coordination Chem Rev 141:23-32). We havefocused on the binding of an Al¹⁸F complex, since aluminum-fluoride canbe relatively stable in vivo (Li, 2003, Crit Rev Oral Biol Med14:100-114; Antonny et al., 1992, J Biol Chem 267:6710-18). Initialstudies showed the feasibility of this approach to prepare an¹⁸F-labeled peptide for in vivo targeting of cancer with a bispecificantibody (bsMAb) pretargeting system, a highly sensitive and specifictechnique for localizing cancer, in some cases better than [¹⁸F]FDG(fluorodeoxyglucose) (McBride et al., 2008, J Nucl Med (suppl) 49:97P;Wagner, 2008, J Nucl Med 49:23N-24N; Karacay et al., 2000, Bioconj Chem11:842-54; Sharkey et al., 2008, Cancer Res 68; 5282-90; Gold Et al.,2008, Cancer Res 68:4819-26; Sharkey et al., 2005, Nature Med11:1250-55; Sharkey et al., 2005, Clin Cancer Res 11:7109s-7121s;McBride et al., 2006, J Nucl Med 47:1678-88; Sharkey et al., 2008,Radiology 246:497-508). These studies revealed that an Al¹⁸F complexcould bind stably to a 1,4,7-triazacyclononane-1,4,7-triacetic acid(NOTA), but the yields were low.

In the Examples below, new labeling conditions and several new chelatingmoieties were examined that enhanced yields from about 10% to about 80%,providing a feasible method for ¹⁸F-labeling of peptides and othermolecules of use in PET imaging.

Targetable Constructs

In certain embodiments, the moiety labeled with ¹⁸F or other diagnosticand/or therapeutic agents may comprise a peptide or other targetableconstruct. Labeled peptides (or proteins), for example RGD peptide,octreotide, bombesin or somatostatin, may be selected to bind directlyto a targeted cell, tissue, pathogenic organism or other target forimaging, detection and/or diagnosis. In other embodiments, labeledpeptides may be selected to bind indirectly, for example using abispecific antibody with one or more binding sites for a targetableconstruct peptide and one or more binding sites for a target antigenassociated with a disease or condition. Bispecific antibodies may beused, for example, in a pretargeting technique wherein the antibody maybe administered first to a subject. Sufficient time may be allowed forthe bispecific antibody to bind to a target antigen and for unboundantibody to clear from circulation. Then a targetable construct, such asa labeled peptide, may be administered to the subject and allowed tobind to the bispecific antibody and localize at the diseased cell ortissue. The distribution of ¹⁸F-labeled targetable constructs may bedetermined by PET scanning or other known techniques.

Such targetable constructs can be of diverse structure and are selectednot only for the availability of an antibody or fragment that binds withhigh affinity to the targetable construct, but also for rapid in vivoclearance when used within the pre-targeting method and bispecificantibodies (bsAb) or multispecific antibodies. Hydrophobic agents arebest at eliciting strong immune responses, whereas hydrophilic agentsare preferred for rapid in vivo clearance. Thus, a balance betweenhydrophobic and hydrophilic character is established. This may beaccomplished, in part, by using hydrophilic chelating agents to offsetthe inherent hydrophobicity of many organic moieties. Also, sub-units ofthe targetable construct may be chosen which have opposite solutionproperties, for example, peptides, which contain amino acids, some ofwhich are hydrophobic and some of which are hydrophilic. Aside frompeptides, carbohydrates may also be used.

Peptides having as few as two amino acid residues, preferably two to tenresidues, may be used and may also be coupled to other moieties, such aschelating agents. The linker should be a low molecular weight conjugate,preferably having a molecular weight of less than 50,000 daltons, andadvantageously less than about 20,000 daltons, 10,000 daltons or 5,000daltons. More usually, the targetable construct peptide will have fouror more residues, such as the peptide DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH₂(SEQ ID NO: 1), wherein DOTA is1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid and HSG is thehistamine succinyl glycyl group. Alternatively, DOTA may be replaced byNOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), TETA(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid), NETA([2-(4,7-biscarboxymethyl[1,4,7]triazacyclononan-1-yl-ethyl]-2-carbonylmethyl-amino]aceticacid) or other known chelating moieties.

The targetable construct may also comprise unnatural amino acids, e.g.,D-amino acids, in the backbone structure to increase the stability ofthe peptide in vivo. In alternative embodiments, other backbonestructures such as those constructed from non-natural amino acids orpeptoids may be used.

The peptides used as targetable constructs are conveniently synthesizedon an automated peptide synthesizer using a solid-phase support andstandard techniques of repetitive orthogonal deprotection and coupling.Free amino groups in the peptide, that are to be used later forconjugation of chelating moieties or other agents, are advantageouslyblocked with standard protecting groups such as a Boc group, whileN-terminal residues may be acetylated to increase serum stability. Suchprotecting groups are well known to the skilled artisan. See Greene andWuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons,N.Y.). When the peptides are prepared for later use within thebispecific antibody system, they are advantageously cleaved from theresins to generate the corresponding C-terminal amides, in order toinhibit in vivo carboxypeptidase activity. Exemplary methods of peptidesynthesis are disclosed in the Examples below.

Where pretargeting with bispecific antibodies is used, the antibody willcontain a first binding site for an antigen produced by or associatedwith a target tissue and a second binding site for a hapten on thetargetable construct. Exemplary haptens include, but are not limited to,HSG and In-DTPA. Antibodies raised to the HSG hapten are known (e.g. 679antibody) and can be easily incorporated into the appropriate bispecificantibody (see, e.g., U.S. Pat. Nos. 6,962,702; 7,138,103 and 7,300,644,incorporated herein by reference with respect to the Examples sections).However, other haptens and antibodies that bind to them are known in theart and may be used, such as In-DTPA and the 734 antibody (e.g., U.S.Pat. No. 7,534,431, the Examples section incorporated herein byreference).

The skilled artisan will realize that although the majority oftargetable constructs disclosed in the Examples below are peptides,other types of molecules may be used as targetable constructs. Forexample, polymeric molecules, such as polyethylene glycol (PEG) may beeasily derivatized with chelating moieties to bind Al¹⁸F. Many examplesof such carrier molecules are known in the art and may be utilized,including but not limited to polymers, nanoparticles, microspheres,liposomes and micelles. For use in pretargeted delivery of ¹⁸F, the onlyrequirement is that the carrier molecule comprise one or more chelatingmoieties for attachment of metal-¹⁸F and one or more hapten moieties tobind to a bispecific or multispecific antibody or other targetingmolecule.

Chelating Moieties

In some embodiments, an ¹⁸F-labeled molecule may comprise one or morehydrophilic chelating moieties, which can bind metal ions and also helpto ensure rapid in vivo clearance. Chelators may be selected for theirparticular metal-binding properties, and may be readily interchanged.

Particularly useful metal-chelate combinations include 2-benzyl-DTPA andits monomethyl and cyclohexyl analogs. Macrocyclic chelators such asNOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), DOTA, TETA(p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid) and NETA arealso of use with a variety of metals, that may potentially be used asligands for ¹⁸F-labeling.

DTPA and DOTA-type chelators, where the ligand includes hard basechelating functions such as carboxylate or amine groups, are mosteffective for chelating hard acid cations, especially Group IIa andGroup IIIa metal cations. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelators such as macrocyclic polyethers are of interest forstably binding nuclides. Porphyrin chelators may be used with numerousmetal complexes. More than one type of chelator may be conjugated to acarrier to bind multiple metal ions. Chelators such as those disclosedin U.S. Pat. No. 5,753,206, especially thiosemicarbazonylglyoxylcysteine(Tscg-Cys) and thiosemicarbazinyl-acetylcysteine (Tsca-Cys) chelatorsare advantageously used to bind soft acid cations of Tc, Re, Bi andother transition metals, lanthanides and actinides that are tightlybound to soft base ligands. It can be useful to link more than one typeof chelator to a peptide. Because antibodies to a di-DTPA hapten areknown (Barbet et al., U.S. Pat. No. 5,256,395) and are readily coupledto a targeting antibody to form a bispecific antibody, it is possible touse a peptide hapten with cold diDTPA chelator and another chelator forbinding an ¹⁸F complex, in a pretargeting protocol. One example of sucha peptide is Ac-Lys(DTPA)-Tyr-Lys(DTPA)-Lys(Tscg-Cys)-NH₂ (core peptidedisclosed as SEQ ID NO:2). Other hard acid chelators such as DOTA, TETAand the like can be substituted for the DTPA and/or Tscg-Cys groups, andMAbs specific to them can be produced using analogous techniques tothose used to generate the anti-di-DTPA MAb.

Another useful chelator may comprise a NOTA-type moiety, for example asdisclosed in Chong et al. (J. Med. Chem., 2008, 51:118-25). Chong et al.disclose the production and use of a bifunctional C-NETA ligand, basedupon the NOTA structure, that when complexed with ¹⁷⁷Lu or ^(205/206)Bishowed stability in serum for up to 14 days. The chelators are notlimiting and these and other examples of chelators that are known in theart and/or described in the following Examples may be used in thepractice of the invention.

It will be appreciated that two different hard acid or soft acidchelators can be incorporated into the targetable construct, e.g., withdifferent chelate ring sizes, to bind preferentially to two differenthard acid or soft acid cations, due to the differing sizes of thecations, the geometries of the chelate rings and the preferred complexion structures of the cations. This will permit two different metals,one or both of which may be attached to ¹⁸F, to be incorporated into atargetable construct for eventual capture by a pretargeted bispecificantibody.

Antibodies

Target Antigens

Targeting antibodies of use may be specific to or selective for avariety of cell surface or disease-associated antigens. Exemplary targetantigens of use for imaging or treating various diseases or conditions,such as a malignant disease, a cardiovascular disease, an infectiousdisease, an inflammatory disease, an autoimmune disease, a metabolicdisease, or a neurological (e.g., neurodegenerative) disease may includeα-fetoprotein (AFP), A3, amyloid beta, CA125, colon-specific antigen-p(CSAp), carbonic anhydrase IX, CCCL19, CCCL21, CSAp, CD1, CD1a, CD2,CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21,CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L,CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74,CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CXCR4,CXCR7, CXCL12, HIF-1α, AFP, CEACAM5, CEACAM6, c-met, B7, ED-B offibronectin, EGP-1, EGP-2, Factor H, FHL-1, fibrin, Flt-3, folatereceptor, glycoprotein IIb/IIIa, GRO-β, human chorionic gonadotropin(HCG), HER-2/neu, HMGB-1, hypoxia inducible factor (HIF), HM1.24,HLA-DR, Ia, ICAM-1, insulin-like growth factor-1 (IGF-1), IGF-1R, IFN-γ,IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-1,IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, KS-1, Le(y),low-density lipoprotein (LDL), MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF,MUC1, MUC2, MUC3, MUC4, MUC5a-c, MUC16, NCA-95, NCA-90, as NF-κB,pancreatic cancer mucin, PAM4 antigen, placental growth factor, p53,PLAGL2, Pr1, prostatic acid phosphatase, PSA, PRAME, PSMA, P1GF,tenascin, RANTES, T101, TAC, TAG72, TF, Tn antigen, Thomson-Friedenreichantigens, thrombin, tumor necrosis antigens, TNF-α, TRAIL receptor (R1and R2), TROP2, VEGFR, EGFR, complement factors C3, C3a, C3b, C5a, C5,and an oncogene product.

In certain embodiments, such as imaging or treating tumors, antibodiesof use may target tumor-associated antigens. These antigenic markers maybe substances produced by a tumor or may be substances which accumulateat a tumor site, on tumor cell surfaces or within tumor cells. Amongsuch tumor-associated markers are those disclosed by Herberman,“Immunodiagnosis of Cancer”, in Fleisher ed., “The Clinical Biochemistryof Cancer”, page 347 (American Association of Clinical Chemists, 1979)and in U.S. Pat. Nos. 4,150,149; 4,361,544; and 4,444,744, the Examplessection of each of which is incorporated herein by reference. Reports ontumor associated antigens (TAAs) include Mizukami et al., (2005, NatureMed. 11:992-97); Hatfield et al., (2005, Curr. Cancer Drug Targets5:229-48); Vallbohmer et al. (2005, J. Clin. Oncol. 23:3536-44); and Renet al. (2005, Ann. Surg. 242:55-63), each incorporated herein byreference with respect to the TAAs identified.

Tumor-associated markers have been categorized by Herberman, supra, in anumber of categories including oncofetal antigens, placental antigens,oncogenic or tumor virus associated antigens, tissue associatedantigens, organ associated antigens, ectopic hormones and normalantigens or variants thereof. Occasionally, a sub-unit of atumor-associated marker is advantageously used to raise antibodieshaving higher tumor-specificity, e.g., the beta-subunit of humanchorionic gonadotropin (HCG) or the gamma region of carcinoembryonicantigen (CEA), which stimulate the production of antibodies having agreatly reduced cross-reactivity to nontumor substances as disclosed inU.S. Pat. Nos. 4,361,644 and 4,444,744.

Another marker of interest is transmembrane activator andCAML-interactor (TACI). See Yu et al. Nat. Immunol. 1:252-256 (2000).Briefly, TACI is a marker for B-cell malignancies (e.g., lymphoma). TACIand B-cell maturation antigen (BCMA) are bound by the tumor necrosisfactor homolog—a proliferation-inducing ligand (APRIL). APRIL stimulatesin vitro proliferation of primary B and T-cells and increases spleenweight due to accumulation of B-cells in vivo. APRIL also competes withTALL-I (also called BLyS or BAFF) for receptor binding. Soluble BCMA andTACI specifically prevent binding of APRIL and block APRIL-stimulatedproliferation of primary B-cells. BCMA-Fc also inhibits production ofantibodies against keyhole limpet hemocyanin and Pneumovax in mice,indicating that APRIL and/or TALL-I signaling via BCMA and/or TACI arerequired for generation of humoral immunity. Thus, APRIL-TALL-I andBCMA-TACI form a two ligand-two receptor pathway involved in stimulationof B and T-cell function.

Where the disease involves a lymphoma, leukemia or autoimmune disorder,targeted antigens may be selected from the group consisting of CD4, CD5,CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38,CD40, CD40L, CD46, CD52, CD54, CD67, CD74, CD79a, CD80, CD126, CD138,CD154, B7, MUC1, Ia, Ii, HM1.24, HLA-DR, tenascin, VEGF, P1GF, ED-Bfibronectin, an oncogene (e.g., c-met or PLAGL2), an oncogene product,CD66a-d, necrosis antigens, IL-2, T101, TAG, IL-6, MIF, TRAIL-R1 (DR4)and TRAIL-R2 (DR5).

In some embodiments, target antigens may be selected from the groupconsisting of (A) proinflammatory effectors of the innate immune system,(B) coagulation factors, (C) complement factors and complementregulatory proteins, and (D) targets specifically associated with aninflammatory or immune-dysregulatory disorder or with a pathologicangiogenesis or cancer, wherein the latter target is not (A), (B), or(C). Suitable targets are described in U.S. patent application Ser. No.11/296,432, filed Dec. 8, 2005, the Examples section of which isincorporated herein by reference.

The proinflammatory effector of the innate immune system may be aproinflammatory effector cytokine, a proinflammatory effector chemokineor a proinflammatory effector receptor. Suitable proinflammatoryeffector cytokines include MIF, HMGB-1 (high mobility group box protein1), TNF-α, IL-1, IL-4, IL-5, IL-6, IL-8, IL-12, IL-15, and IL-18.Examples of proinflammatory effector chemokines include CCL19, CCL21,IL-8, MCP-1, RANTES, MIP-1A, MIP-1B, ENA-78, MCP-1, IP-10, GRO-β, andeotaxin. Proinflammatory effector receptors include IL-4R (interleukin-4receptor), IL-6R (interleukin-6 receptor), IL-13R (interleukin-13receptor), IL-15R (interleukin-15 receptor) and IL-18R (interleukin-18receptor).

The targeting molecule may bind to a coagulation factor, such as tissuefactor (TF) or thrombin. In other embodiments, the targeting moleculemay bind to a complement factor or complement regulatory protein. Inpreferred embodiments, the complement factor is selected from the groupconsisting of C3, C5, C3a, C3b, and C5a. When the targeting moleculebinds to a complement regulatory protein, the complement regulatoryprotein preferably is selected from the group consisting of CD46, CD55,CD59 and mCRP.

MIF is a pivotal cytokine of the innate immune system and plays animportant part in the control of inflammatory responses. Originallydescribed as a T lymphocyte-derived factor that inhibited the randommigration of macrophages, the protein known as macrophage migrationinhibitory factor (MIF) was an enigmatic cytokine for almost 3 decades.In recent years, the discovery of MIF as a product of the anteriorpituitary gland and the cloning and expression of bioactive, recombinantMIF protein have led to the definition of its critical biological rolein vivo. MIF has the unique property of being released from macrophagesand T lymphocytes that have been stimulated by glucocorticoids. Oncereleased, MIF overcomes the inhibitory effects of glucocorticoids onTNF-α, IL-1β, IL-6, and IL-8 production by LPS-stimulated monocytes invitro and suppresses the protective effects of steroids against lethalendotoxemia in vivo. MIF also antagonizes glucocorticoid inhibition ofT-cell proliferation in vitro by restoring IL-2 and IFN-gammaproduction. MIF is the first mediator to be identified that cancounter-regulate the inhibitory effects of glucocorticoids and thusplays a critical role in the host control of inflammation and immunity.MIF is particularly of use in cancer, pathological angiogenesis, andsepsis or septic shock. More recently, CD74 has been identified as anendogenous receptor for MIF, along with CD44, CXCR2 and CXCR4 (see,e.g., Baron et al., 2011, J Neuroscience Res 89:711-17). Targetingmolecules that bind to MIF, CD74, CD44, CXCR2 and/or CXCR4 may be of usefor imaging various of these conditions.

HMGB-1, a DNA binding nuclear and cytosolic protein, is aproinflammatory cytokine released by monocytes and macrophages that havebeen activated by IL-1β, TNF, or LPS. Via its B box domain, it inducesphenotypic maturation of DCs. It also causes increased secretion of theproinflammatory cytokines IL-1α, IL-6, IL-8, IL-12, TNF-α and RANTES.HMGB-1 released by necrotic cells may be a signal of tissue or cellularinjury that, when sensed by DCs, induces and/or enhances an immunereaction. Palumbo et al. report that HMBG1 induces mesoangioblastmigration and proliferation (J Cell Biol, 164:441-449, 2004). Targetingmolecules that target HMBG-1 may be of use in detecting, diagnosing ortreating arthritis, particularly collagen-induced arthritis, sepsisand/or septic shock. Yang et al., PNAS USA 101:296-301 (2004); Kokkolaet al., Arthritis Rheum, 48:2052-8 (2003); Czura et al., J Infect Dis,187 Suppl 2:S391-6 (2003); Treutiger et al., J Intern Med, 254:375-85(2003).

TNF-α is an important cytokine involved in systemic inflammation and theacute phase response. TNF-α is released by stimulated monocytes,fibroblasts, and endothelial cells. Macrophages, T-cells andB-lymphocytes, granulocytes, smooth muscle cells, eosinophils,chondrocytes, osteoblasts, mast cells, glial cells, and keratinocytesalso produce TNF-α after stimulation. Its release is stimulated byseveral other mediators, such as interleukin-1 and bacterial endotoxin,in the course of damage, e.g., by infection. It has a number of actionson various organ systems, generally together with interleukins-1 and -6.TNF-α is a useful target for sepsis or septic shock.

The complement system is a complex cascade involving proteolyticcleavage of serum glycoproteins often activated by cell receptors. The“complement cascade” is constitutive and non-specific but it must beactivated in order to function. Complement activation results in aunidirectional sequence of enzymatic and biochemical reactions. In thiscascade, a specific complement protein, C5, forms two highly active,inflammatory byproducts, C5a and C5b, which jointly activate white bloodcells. This in turn evokes a number of other inflammatory byproducts,including injurious cytokines, inflammatory enzymes, and cell adhesionmolecules. Together, these byproducts can lead to the destruction oftissue seen in many inflammatory diseases. This cascade ultimatelyresults in induction of the inflammatory response, phagocyte chemotaxisand opsonization, and cell lysis.

The complement system can be activated via two distinct pathways, theclassical pathway and the alternate pathway. Some of the components mustbe enzymatically cleaved to activate their function; others simplycombine to form complexes that are active. Active components of theclassical pathway include C1q, C1r, C1s, C2a, C2b, C3a, C3b, C4a, andC4b. Active components of the alternate pathway include C3a, C3b, FactorB, Factor Ba, Factor Bb, Factor D, and Properdin. The last stage of eachpathway is the same, and involves component assembly into a membraneattack complex. Active components of the membrane attack complex includeC5a, C5b, C6, C7, C8, and C9n.

While any of these components of the complement system can be targeted,certain of the complement components are preferred. C3a, C4a and C5acause mast cells to release chemotactic factors such as histamine andserotonin, which attract phagocytes, antibodies and complement, etc.These form one group of preferred targets. Another group of preferredtargets includes C3b, C4b and C5b, which enhance phagocytosis of foreigncells. Another preferred group of targets are the predecessor componentsfor these two groups, i.e., C3, C4 and C5. C5b, C6, C7, C8 and C9 inducelysis of foreign cells (membrane attack complex) and form yet anotherpreferred group of targets.

Coagulation factors also are preferred targets, particularly tissuefactor (TF) and thrombin. TF is also known also as tissuethromboplastin, CD142, coagulation factor III, or factor III. TF is anintegral membrane receptor glycoprotein and a member of the cytokinereceptor superfamily. The ligand binding extracellular domain of TFconsists of two structural modules with features that are consistentwith the classification of TF as a member of type-2 cytokine receptors.TF is involved in the blood coagulation protease cascade and initiatesboth the extrinsic and intrinsic blood coagulation cascades by forminghigh affinity complexes between the extracellular domain of TF and thecirculating blood coagulation factors, serine proteases factor VII orfactor VIIa. These enzymatically active complexes then activate factorIX and factor X, leading to thrombin generation and clot formation.

TF is expressed by various cell types, including monocytes, macrophagesand vascular endothelial cells, and is induced by IL-1, TNF-α orbacterial lipopolysaccharides. Protein kinase C is involved in cytokineactivation of endothelial cell TF expression. Induction of TF byendotoxin and cytokines is an important mechanism for initiation ofdisseminated intravascular coagulation seen in patients withGram-negative sepsis. TF also appears to be involved in a variety ofnon-hemostatic functions including inflammation, cancer, brain function,immune response, and tumor-associated angiogenesis. Thus, targetingmolecules that target TF are of use in coagulopathies, sepsis, cancer,pathologic angiogenesis, and other immune and inflammatory dysregulatorydiseases.

In other embodiments, the targeting molecule may bind to a MHC class I,MHC class II or accessory molecule, such as CD40, CD54, CD80 or CD86.The binding molecule also may bind to a T-cell activation cytokine, orto a cytokine mediator, such as NF-κB. Targets associated with sepsisand immune dysregulation and other immune disorders include MIF, IL-1,IL-6, IL-8, CD74, CD83, and C5aR. Antibodies and inhibitors against C5aRhave been found to improve survival in rodents with sepsis (Huber-Langet al., FASEB J 2002; 16:1567-1574; Riedemann et al., J Clin Invest2002; 110:101-108) and septic shock and adult respiratory distresssyndrome in monkeys (Hangen et al., J Surg Res 1989; 46:195-199; Stevenset al., J Clin Invest 1986; 77:1812-1816). Thus, for sepsis, preferredtargets are associated with infection, such as LPS/C5a. Other preferredtargets include HMGB-1, TF, CD14, VEGF, and IL-6, each of which isassociated with septicemia or septic shock.

In still other embodiments, a target may be associated with graft versushost disease or transplant rejection, such as MIF (Lo et al., BoneMarrow Transplant, 30(6):375-80 (2002)), CD74 or HLA-DR. A target alsomay be associated with acute respiratory distress syndrome, such as IL-8(Bouros et al., PMC Pulm Med, 4(1):6 (2004), atherosclerosis orrestenosis, such as MIF (Chen et al., Arterioscler Thromb Vasc Biol,24(4):709-14 (2004), asthma, such as IL-18 (Hata et al., Int Immunol,Oct. 11, 2004 Epub ahead of print), a granulomatous disease, such asTNF-α (Ulbricht et al., Arthritis Rheum, 50(8):2717-8 (2004), aneuropathy, such as carbamylated EPO (erythropoietin) (Leist et al.,Science 305(5681):164-5 (2004), or cachexia, such as IL-6 and TNF-α.

Other targets include C5a, LPS, IFN-gamma, B7; CD2, CD4, CD14, CD18,CD11a, CD11b, CD11c, CD14, CD18, CD27, CD29, CD38, CD40L, CD52, CD64,CD83, CD147, CD154. Activation of mononuclear cells by certain microbialantigens, including LPS, can be inhibited to some extent by antibodiesto CD 18, CD 11b, or CD 11 c, which thus implicate β₂ integrins (Cuzzolaet al., J Immunol 2000; 164:5871-5876; Medvedev et al., J Immunol 1998;160: 4535-4542). CD83 has been found to play a role in giant cellarteritis (GCA), which is a systemic vasculitis that affects medium- andlarge-size arteries, predominately the extracranial branches of theaortic arch and of the aorta itself, resulting in vascular stenosis andsubsequent tissue ischemia, and the severe complications of blindness,stroke and aortic arch syndrome (Weyand and Goronzy, N Engl J Med 2003;349:160-169; Hunder and Valente, In: Inflammatory Diseases of BloodVessels. G. S. Hoffman and C. M. Weyand, eds, Marcel Dekker, New York,2002; 255-265). Antibodies to CD83 were found to abrogate vasculitis ina SCID mouse model of human GCA (Ma-Krupa et al., J Exp Med 2004;199:173-183), suggesting to these investigators that dendritic cells,which express CD83 when activated, are critical antigen-processing cellsin GCA. In these studies, they used a mouse anti-CD83 MAb (IgG1 cloneHB15e from Research Diagnostics). CD154, a member of the TNF family, isexpressed on the surface of CD4-positive T-lymphocytes, and it has beenreported that a humanized monoclonal antibody to CD154 producedsignificant clinical benefit in patients with active systemic lupuserythematosus (SLE) (Grammar et al., J Clin Invest 2003; 112:1506-1520).It also suggests that this antibody might be useful in other autoimmunediseases (Kelsoe, J Clin Invest 2003; 112:1480-1482). Indeed, thisantibody was also reported as effective in patients with refractoryimmune thrombocytopenic purpura (Kuwana et al., Blood 2004;103:1229-1236).

In rheumatoid arthritis, a recombinant interleukin-1 receptorantagonist, IL-1 Ra or anakinra, has shown activity (Cohen et al., AnnRheum Dis 2004; 63:1062-8; Cohen, Rheum Dis Clin North Am 2004;30:365-80). An improvement in treatment of these patients, whichhitherto required concomitant treatment with methotrexate, is to combineanakinra with one or more of the anti-proinflammatory effector cytokinesor anti-proinflammatory effector chemokines (as listed above). Indeed,in a review of antibody therapy for rheumatoid arthritis, Taylor (CurrOpin Pharmacol 2003; 3:323-328) suggests that in addition to TNF, otherantibodies to such cytokines as IL-1, IL-6, IL-8, IL-15, IL-17 andIL-18, are useful.

Methods for Raising Antibodies

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A or Protein-G Sepharose,size-exclusion chromatography, and ion-exchange chromatography. See, forexample, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, seeBaines et al., “Purification of Immunoglobulin G (IgG),” in METHODS INMOLECULAR BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art, as discussedbelow.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 6: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(κ) and V_(H)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman κ and IgG₁ constant region domains.

Humanized Antibodies

Techniques for producing humanized MAbs are well known in the art (see,e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter etal., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev.Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150:2844 (1993)).A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Preferred residues forsubstitution include FR residues that are located within 1, 2, or 3Angstroms of a CDR residue side chain, that are located adjacent to aCDR sequence, or that are predicted to interact with a CDR residue.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Pharmacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art. Phage display can be performed ina variety of formats, for their review, see e.g. Johnson and Chiswell,Current Opinion in Structural Biology 3:5564-571 (1993).

Human antibodies may also be generated by in vitro activated B-cells.See U.S. Pat. Nos. 5,567,610 and 5,229,275, incorporated herein byreference in their entirety. The skilled artisan will realize that thesetechniques are exemplary and any known method for making and screeninghuman antibodies or antibody fragments may be utilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXenoMouse® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23,incorporated herein by reference) from Abgenix (Fremont, Calif.). In theXenoMouse® and similar animals, the mouse antibody genes have beeninactivated and replaced by functional human antibody genes, while theremainder of the mouse immune system remains intact.

The XenoMouse® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along with accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B-cells,which may be processed into hybridomas by known techniques. A XenoMouse®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XenoMouse®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XenoMouse® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Known Antibodies

The skilled artisan will realize that the targeting molecules of use forimaging, detection and/or diagnosis may incorporate any antibody orfragment known in the art that has binding specificity for a targetantigen associated with a disease state or condition. Such knownantibodies include, but are not limited to, hR1 (anti-IGF-1R, U.S.patent application Ser. No. 12/772,645, filed Mar. 12, 2010) hPAM4(anti-pancreatic cancer mucin, U.S. Pat. No. 7,282,567), hA20(anti-CD20, U.S. Pat. No. 7,251,164), hA19 (anti-CD19, U.S. Pat. No.7,109,304), hIMMU31 (anti-AFP, U.S. Pat. No. 7,300,655), hLL1(anti-CD74, U.S. Pat. No. 7,312,318), hLL2 (anti-CD22, U.S. Pat. No.7,074,403), hMu-9 (anti-CSAp, U.S. Pat. No. 7,387,773), hL243(anti-HLA-DR, U.S. Pat. No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Pat.No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Pat. No. 7,662,378, U.S.patent application Ser. No. 12/846,062, filed Jul. 29, 2010), hRS7(anti-TROP2), U.S. Pat. No. 7,238,785), hMN-3 (anti-CEACAM6, U.S. Pat.No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Pat. No. 7,138,496)the Examples section of each cited patent or application incorporatedherein by reference.

Alternative antibodies of use include, but are not limited to, abciximab(anti-glycoprotein alemtuzumab (anti-CD52), bevacizumab (anti-VEGF),cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan(anti-CD20), panitumumab (anti-EGFR), rituximab (anti-CD20), tositumomab(anti-CD20), trastuzumab (anti-ErbB2), abagovomab (anti-CA-125),adecatumumab (anti-EpCAM), atlizumab (anti-IL-6 receptor), benralizumab(anti-CD125), CC49 (anti-TAG-72), AB-PG1-XG1-026 (anti-PSMA, U.S. patentapplication Ser. No. 11/983,372, deposited as ATCC PTA-4405 andPTA-4406), D2/B (anti-PSMA, WO 2009/130575), tocilizumab (anti-IL-6receptor), basiliximab (anti-CD25), daclizumab (anti-CD25), efalizumab(anti-CD11a), GA101 (anti-CD20; Glycart Roche), muromonab-CD3 (anti-CD3receptor), natalizumab (anti-α4 integrin), omalizumab (anti-IgE);anti-TNF-α antibodies such as CDP571 (Ofei et al., 2011, Diabetes45:881-85), MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B, M303 (ThermoScientific, Rockford, Ill.), infliximab (CENTOCOR, Malvern, Pa.),certolizumab pegol (UCB, Brussels, Belgium), anti-CD70L (UCB, Brussels,Belgium), adalimumab (Abbott, Abbott Park, Ill.), Benlysta (Human GenomeSciences); and antibodies against pathogens such as CR6261(anti-influenza), exbivirumab (anti-hepatitis B), felvizumab(anti-respiratory syncytial virus), foravirumab (anti-rabies virus),motavizumab (anti-respiratory syncytial virus), palivizumab(anti-respiratory syncytial virus), panobacumab (anti-Pseudomonas),rafivirumab (anti-rabies virus), regavirumab (anti-cytomegalovirus),sevirumab (anti-cytomegalovirus), tivirumab (anti-hepatitis B), andurtoxazumab (anti-E. coli).

Other antibodies are known to target antigens associated with diseasedcells, tissues or organs. For example, bapineuzumab is in clinicaltrials for therapy of Alzheimer's disease. Other antibodies proposed forAlzheimer's disease include Alz 50 (Ksiezak-Reding et al., 1987, J BiolChem 263:7943-47), gantenerumab, and solanezumab. Anti-CD3 antibodieshave been proposed for type 1 diabetes (Cernea et al., 2010, DiabetesMetab Rev 26:602-05). Antibodies to fibrin (e.g., scFv(59D8); T2G1s;MH1) are known and in clinical trials as imaging agents for disclosingfibrin clots and pulmonary emboli, while anti-granulocyte antibodies,such as MN-3, MN-15, anti-NCA95, and anti-CD15 antibodies, can targetmyocardial infarcts and myocardial ischemia. (See, e.g., U.S. Pat. Nos.5,487,892; 5,632,968; 6,294,173; 7,541,440, the Examples section of eachincorporated herein by reference) Anti-macrophage, anti-low-densitylipoprotein (LDL) and anti-CD74 (e.g., hLL1) antibodies can be used totarget atherosclerotic plaques. Abciximab (anti-glycoprotein IIb/IIIa)has been approved for adjuvant use for prevention of restenosis inpercutaneous coronary interventions and the treatment of unstable angina(Waldmann et al., 2000, Hematol 1:394-408). Anti-CD3 antibodies havebeen reported to reduce development and progression of atherosclerosis(Steffens et al., 2006, Circulation 114:1977-84). Antibodies againstoxidized LDL induced a regression of established atherosclerosis in amouse model (Ginsberg, 2007, J Am Coll Cardiol 52:2319-21). Anti-ICAM-1antibody was shown to reduce ischemic cell damage after cerebral arteryocclusion in rats (Zhang et al., 1994, Neurology 44:1747-51).Commercially available monoclonal antibodies to leukocyte antigens arerepresented by: OKT anti-T cell monoclonal antibodies (available fromOrtho Pharmaceutical Company) which bind to normal T-lymphocytes; themonoclonal antibodies produced by the hybridomas having the ATCCaccession numbers HB44, HB55, HB12, HB78 and HB2; G7E11, W8E7, NKP15 andG022 (Becton Dickinson); NEN9.4 (New England Nuclear); and FMC11 (SeraLabs). A description of antibodies against fibrin and platelet antigensis contained in Knight, Semin. Nucl. Med., 20:52-67 (1990).

Known antibodies of use may bind to antigens produced by or associatedwith pathogens, such as HIV. Such antibodies may be used to detect,diagnose and/or treat infectious disease. Candidate anti-HIV antibodiesinclude the anti-envelope antibody described by Johansson et al. (AIDS.2006 Oct. 3; 20(15):1911-5), as well as the anti-HIV antibodiesdescribed and sold by Polymun (Vienna, Austria), also described in U.S.Pat. No. 5,831,034, U.S. Pat. No. 5,911,989, and Vcelar et al., AIDS2007; 21(16):2161-2170 and Joos et al., Antimicrob. Agents Chemother.2006; 50(5):1773-9, all incorporated herein by reference.

Antibodies against malaria parasites can be directed against thesporozoite, merozoite, schizont and gametocyte stages. Monoclonalantibodies have been generated against sporozoites (circumsporozoiteantigen), and have been shown to bind to sporozoites in vitro and inrodents (N. Yoshida et al., Science 207:71-73, 1980). Several groupshave developed antibodies to T. gondii, the protozoan parasite involvedin toxoplasmosis (Kasper et al., J. Immunol. 129:1694-1699, 1982; Id.,30:2407-2412, 1983). Antibodies have been developed againstschistosomular surface antigens and have been found to bind toschistosomulae in vivo or in vitro (Simpson et al., Parasitology,83:163-177, 1981; Smith et al., Parasitology, 84:83-91, 1982: Gryzch etal., J. Immunol., 129:2739-2743, 1982; Zodda et al., J. Immunol.129:2326-2328, 1982; Dissous et al., J. Immunol., 129:2232-2234, 1982)

Trypanosoma cruzi is the causative agent of Chagas' disease, and istransmitted by blood-sucking reduviid insects. An antibody has beengenerated that specifically inhibits the differentiation of one form ofthe parasite to another (epimastigote to trypomastigote stage) in vitroand which reacts with a cell-surface glycoprotein; however, this antigenis absent from the mammalian (bloodstream) forms of the parasite (Sheret al., Nature, 300:639-640, 1982).

Anti-fungal antibodies are known in the art, such as anti-Sclerotiniaantibody (U.S. Pat. No. 7,910,702); antiglucuronoxylomannan antibody(Zhong and Priofski, 1998, Clin Diag Lab Immunol 5:58-64); anti-Candidaantibodies (Matthews and Burnie, 2001, 2:472-76); andanti-glycosphingolipid antibodies (Toledo et al., 2010, BMC Microbiol10:47).

Where bispecific antibodies are used, the second MAb may be selectedfrom any anti-hapten antibody known in the art, including but notlimited to h679 (U.S. Pat. No. 7,429,381) and 734 (U.S. Pat. Nos.7,429,381; 7,563,439; 7,666,415; and 7,534,431), the Examples section ofeach of which is incorporated herein by reference.

Various other antibodies of use are known in the art (e.g., U.S. Pat.Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104;6,730.300; 6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084;7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318;7,585,491; 7,612,180; 7,642,239 and U.S. Patent Application Publ. No.20060193865; each incorporated herein by reference.) Such knownantibodies are of use for detection and/or imaging of a variety ofdisease states or conditions (e.g., hMN-14 or TF2 (CEA-expressingcarcinomas), hA20 or TF-4 (lymphoma), hPAM4 or TF-10 (pancreaticcancer), RS7 (lung, breast, ovarian, prostatic cancers), hMN-15 or hMN3(inflammation), anti-gp120 and/or anti-gp41 (HIV), anti-platelet andanti-thrombin (clot imaging), anti-myosin (cardiac necrosis), anti-CXCR4(cancer and inflammatory disease)).

Antibodies of use may be commercially obtained from a wide variety ofknown sources. For example, a variety of antibody secreting hybridomalines are available from the American Type Culture Collection (ATCC,Manassas, Va.). A large number of antibodies against various diseasetargets, including but not limited to tumor-associated antigens, havebeen deposited at the ATCC and/or have published variable regionsequences and are available for use in the claimed methods andcompositions. See, e.g., U.S. Pat. Nos. 7,312,318; 7,282,567; 7,151,164;7,074,403; 7,060,802; 7,056,509; 7,049,060; 7,045,132; 7,041,803;7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133; 7,001,598;6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018;6,964,854; 6,962,981; 6,962,813; 6,956,107; 6,951,924; 6,949,244;6,946,129; 6,943,020; 6,939,547; 6,921,645; 6,921,645; 6,921,533;6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879; 6,893,625;6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580;6,872,568; 6,867,006; 6,864,062; 6,861,511; 6,861,227; 6,861,226;6,838,282; 6,835,549; 6,835,370; 6,824,780; 6,824,778; 6,812,206;6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688; 6,764,681;6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966;6,709,653; 6,693,176; 6,692,908; 6,689,607; 6,689,362; 6,689,355;6,682,737; 6,682,736; 6,682,734; 6,673,344; 6,653,104; 6,652,852;6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441; 6,605,279;6,596,852; 6,592,868; 6,576,745; 6,572,856; 6,566,076; 6,562,618;6,545,130; 6,544,749; 6,534,058; 6,528,625; 6,528,269; 6,521,227;6,518,404; 6,511,665; 6,491,915; 6,488,930; 6,482,598; 6,482,408;6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823; 6,458,356;6,455,044; 6,455,040, 6,451,310; 6,444,206; 6,441,143; 6,432,404;6,432,402; 6,419,928; 6,413,726; 6,406,694; 6,403,770; 6,403,091;6,395,276; 6,395,274; 6,387,350; 6,383,759; 6,383,484; 6,376,654;6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245; 6,355,244;6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393;6,254,868; 6,187,287; 6,183,744; 6,129,914; 6,120,767; 6,096,289;6,077,499; 5,922,302; 5,874,540; 5,814,440; 5,798,229; 5,789,554;5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459; 5,443,953,5,525,338. These are exemplary only and a wide variety of otherantibodies and their hybridomas are known in the art. The skilledartisan will realize that antibody sequences or antibody-secretinghybridomas against almost any disease-associated antigen may be obtainedby a simple search of the ATCC, NCBI and/or USPTO databases forantibodies against a selected disease-associated target of interest. Theantigen binding domains of the cloned antibodies may be amplified,excised, ligated into an expression vector, transfected into an adaptedhost cell and used for protein production, using standard techniqueswell known in the art.

Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. The antibody fragments are antigen binding portions ofan antibody, such as F(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv and the like.F(ab′)₂ fragments can be produced by pepsin digestion of the antibodymolecule and Fab′ fragments can be generated by reducing disulfidebridges of the F(ab′)₂ fragments. Alternatively, Fab′ expressionlibraries can be constructed (Huse et al., 1989, Science, 246:1274-1281)to allow rapid and easy identification of monoclonal Fab′ fragments withthe desired specificity. An antibody fragment can be prepared byproteolytic hydrolysis of the full length antibody or by expression inE. coli or another host of the DNA coding for the fragment. Thesemethods are described, for example, by Goldenberg, U.S. Pat. Nos.4,036,945 and 4,331,647 and references contained therein, which patentsare incorporated herein in their entireties by reference. Also, seeNisonoff et al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem.J. 73: 119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

A single chain Fv molecule (scFv) comprises a V_(L) domain and a V_(H)domain. The V_(L) and V_(H) domains associate to form a target bindingsite. These two domains are further covalently linked by a peptidelinker (L). Methods for making scFv molecules and designing suitablepeptide linkers are described in U.S. Pat. No. 4,704,692, U.S. Pat. No.4,946,778, R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80(1995) and R. E. Bird and B. W. Walker, “Single Chain Antibody VariableRegions,” TIBTECH, Vol 9: 132-137 (1991), incorporated herein byreference.

An scFv library with a large repertoire can be constructed by isolatingV-genes from non-immunized human donors using PCR primers correspondingto all known V_(H), V_(kappa) and V₈₀ gene families. See, e.g., Vaughnet al., Nat. Biotechnol., 14: 309-314 (1996). Following amplification,the V_(kappa) and V_(lambda), pools are combined to form one pool. Thesefragments are ligated into a phagemid vector. The scFv linker is thenligated into the phagemid upstream of the V_(L) fragment. The V_(H) andlinker-V_(L) fragments are amplified and assembled on the J_(H) region.The resulting V_(H)-linker-V_(L) fragments are ligated into a phagemidvector. The phagemid library can be panned for binding to the selectedantigen.

Other antibody fragments, for example single domain antibody fragments,are known in the art and may be used in the claimed constructs. Singledomain antibodies (VHH) may be obtained, for example, from camels,alpacas or llamas by standard immunization techniques. (See, e.g.,Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods281:161-75, 2003; Maass et al., J Immunol Methods 324:13-25, 2007). TheVHH may have potent antigen-binding capacity and can interact with novelepitopes that are inaccessible to conventional VH-VL pairs. (Muyldermanset al., 2001) Alpaca serum IgG contains about 50% camelid heavy chainonly IgG antibodies (Cabs) (Maass et al., 2007). Alpacas may beimmunized with known antigens and VHHs can be isolated that bind to andneutralize the target antigen (Maass et al., 2007). PCR primers thatamplify virtually all alpaca VHH coding sequences have been identifiedand may be used to construct alpaca VHH phage display libraries, whichcan be used for antibody fragment isolation by standard biopanningtechniques well known in the art (Maass et al., 2007). These and otherknown antigen-binding antibody fragments may be utilized in the claimedmethods and compositions.

General Techniques for Antibody Cloning and Production

Various techniques, such as production of chimeric or humanizedantibodies, may involve procedures of antibody cloning and construction.The antigen-binding Vκ (variable light chain) and V_(H) (variable heavychain) sequences for an antibody of interest may be obtained by avariety of molecular cloning procedures, such as RT-PCR, 5′-RACE, andcDNA library screening. The V genes of a MAb from a cell that expressesa murine MAb can be cloned by PCR amplification and sequenced. Toconfirm their authenticity, the cloned V_(L) and V_(H) genes can beexpressed in cell culture as a chimeric Ab as described by Orlandi etal., (Proc. Natl. Acad. Sci., USA, 86: 3833 (1989)). Based on the V genesequences, a humanized MAb can then be designed and constructed asdescribed by Leung et al. (Mol. Immunol., 32: 1413 (1995)).

cDNA can be prepared from any known hybridoma line or transfected cellline producing a murine MAb by general molecular cloning techniques(Sambrook et al., Molecular Cloning, A laboratory manual, 2^(nd) Ed(1989)). The Vκ sequence for the MAb may be amplified using the primersVK1BACK and VK1FOR (Orlandi et al., 1989) or the extended primer setdescribed by Leung et al. (BioTechniques, 15: 286 (1993)). The V_(H)sequences can be amplified using the primer pair VH1BACK/VH1FOR (Orlandiet al., 1989) or the primers annealing to the constant region of murineIgG described by Leung et al. (Hybridoma, 13:469 (1994)). Humanized Vgenes can be constructed by a combination of long oligonucleotidetemplate syntheses and PCR amplification as described by Leung et al.(Mol. Immunol., 32: 1413 (1995)).

PCR products for Vκ can be subcloned into a staging vector, such as apBR327-based staging vector, VKpBR, that contains an Ig promoter, asignal peptide sequence and convenient restriction sites. PCR productsfor V_(H) can be subcloned into a similar staging vector, such as thepBluescript-based VHpBS. Expression cassettes containing the Vκ andV_(H) sequences together with the promoter and signal peptide sequencescan be excised from VKpBR and VHpBS and ligated into appropriateexpression vectors, such as pKh and pG1g, respectively (Leung et al.,Hybridoma, 13:469 (1994)). The expression vectors can be co-transfectedinto an appropriate cell and supernatant fluids monitored for productionof a chimeric, humanized or human MAb. Alternatively, the Vκ and V_(H)expression cassettes can be excised and subcloned into a singleexpression vector, such as pdHL2, as described by Gillies et al. (J.Immunol. Methods 125:191 (1989) and also shown in Losman et al., Cancer,80:2660 (1997)).

In an alternative embodiment, expression vectors may be transfected intohost cells that have been pre-adapted for transfection, growth andexpression in serum-free medium. Exemplary cell lines that may be usedinclude the Sp/EEE, Sp/ESF and Sp/ESF-X cell lines (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930 and 7,608,425; the Examples section of each ofwhich is incorporated herein by reference). These exemplary cell linesare based on the Sp2/0 myeloma cell line, transfected with a mutantBcl-EEE gene, exposed to methotrexate to amplify transfected genesequences and pre-adapted to serum-free cell line for proteinexpression.

Bispecific and Multispecific Antibodies

Certain embodiments concern pretargeting methods with bispecificantibodies and hapten-bearing targetable constructs. Numerous methods toproduce bispecific or multispecific antibodies are known, as disclosed,for example, in U.S. Pat. No. 7,405,320, the Examples section of whichis incorporated herein by reference. Bispecific antibodies can beproduced by the quadroma method, which involves the fusion of twodifferent hybridomas, each producing a monoclonal antibody recognizing adifferent antigenic site (Milstein and Cuello, Nature, 1983;305:537-540).

Another method for producing bispecific antibodies usesheterobifunctional cross-linkers to chemically tether two differentmonoclonal antibodies (Staerz, et al. Nature. 1985; 314:628-631; Perez,et al. Nature. 1985; 316:354-356). Bispecific antibodies can also beproduced by reduction of each of two parental monoclonal antibodies tothe respective half molecules, which are then mixed and allowed toreoxidize to obtain the hybrid structure (Staerz and Bevan. Proc NatlAcad Sci USA. 1986; 83:1453-1457). Other methods include improving theefficiency of generating hybrid hybridomas by gene transfer of distinctselectable markers via retrovirus-derived shuttle vectors intorespective parental hybridomas, which are fused subsequently (DeMonte,et al. Proc Natl Acad Sci USA. 1990, 87:2941-2945); or transfection of ahybridoma cell line with expression plasmids containing the heavy andlight chain genes of a different antibody.

Cognate V_(H) and V_(L) domains can be joined with a peptide linker ofappropriate composition and length (usually consisting of more than 12amino acid residues) to form a single-chain Fv (scFv), as discussedabove. Reduction of the peptide linker length to less than 12 amino acidresidues prevents pairing of V_(H) and V_(L) domains on the same chainand forces pairing of V_(H) and V_(L) domains with complementary domainson other chains, resulting in the formation of functional multimers.Polypeptide chains of V_(H) and V_(L) domains that are joined withlinkers between 3 and 12 amino acid residues form predominantly dimers(termed diabodies). With linkers between 0 and 2 amino acid residues,trimers (termed triabody) and tetramers (termed tetrabody) are favored,but the exact patterns of oligomerization appear to depend on thecomposition as well as the orientation of V-domains (V_(H)-linker-V_(L)or V_(L)-linker-V_(H)), in addition to the linker length.

These techniques for producing multispecific or bispecific antibodiesexhibit various difficulties in terms of low yield, necessity forpurification, low stability or the labor-intensiveness of the technique.More recently, a technique known as “dock and lock” (DNL), discussed inmore detail below, has been utilized to produce combinations ofvirtually any desired antibodies, antibody fragments and other effectormolecules (see, e.g., U.S. Patent Application Publ. Nos. 20060228357;20060228300; 20070086942; 20070140966 and 20070264265, the Examplessection of each incorporated herein by reference). The DNL techniqueallows the assembly of monospecific, bispecific or multispecificantibodies, either as naked antibody moieties or in combination with awide range of other effector molecules such as immunomodulators,enzymes, chemotherapeutic agents, chemokines, cytokines, diagnosticagents, therapeutic agents, radionuclides, imaging agents,anti-angiogenic agents, growth factors, oligonucleotides, siderophores,hormones, peptides, toxins, pro-apoptotic agents, or a combinationthereof. Any of the techniques known in the art for making bispecific ormultispecific antibodies may be utilized in the practice of thepresently claimed methods.

Dock-and-Lock (DNL)

In preferred embodiments, bispecific or multispecific antibodies orother constructs may be produced using the dock-and-lock technology(see, e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787and 7,666,400, the Examples section of each incorporated herein byreference). The DNL method exploits specific protein/proteininteractions that occur between the regulatory (R) subunits ofcAMP-dependent protein kinase (PKA) and the anchoring domain (AD) ofA-kinase anchoring proteins (AKAPs) (Baillie et al., FEBS Letters. 2005;579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004; 5: 959). PKA,which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has a and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). The Rsubunits have been isolated only as stable dimers and the dimerizationdomain has been shown to consist of the first 44 amino-terminal residues(Newlon et al., Nat. Struct. Biol. 1999; 6:222). Binding of cAMP to theR subunits leads to the release of active catalytic subunits for a broadspectrum of serine/threonine kinase activities, which are orientedtoward selected substrates through the compartmentalization of PKA viaits docking with AKAPs (Scott et al., J. Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human RIIαand the AD of AKAP as an excellent pair of linker modules for dockingany two entities, referred to hereafter as A and B, into a noncovalentcomplex, which could be further locked into a binding molecule throughthe introduction of cysteine residues into both the DDD and AD atstrategic positions to facilitate the formation of disulfide bonds. Thegeneral methodology of the “dock-and-lock” approach is as follows.Entity A is constructed by linking a DDD sequence to a precursor of A,resulting in a first component hereafter referred to as a. Because theDDD sequence would effect the spontaneous formation of a dimer, A wouldthus be composed of a₂. Entity B is constructed by linking an ADsequence to a precursor of B, resulting in a second component hereafterreferred to as b. The dimeric motif of DDD contained in a₂ will create adocking site for binding to the AD sequence contained in b, thusfacilitating a ready association of a₂ and b to form a binary, trimericcomplex composed of a₂b. This binding event is made irreversible with asubsequent reaction to covalently secure the two entities via disulfidebridges, which occurs very efficiently based on the principle ofeffective local concentration because the initial binding interactionsshould bring the reactive thiol groups placed onto both the DDD and ADinto proximity (Chmura et al., Proc. Natl. Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations oflinkers, adaptor modules and precursors, a wide variety of DNLconstructs of different stoichiometry may be produced and used,including but not limited to dimeric, trimeric, tetrameric, pentamericand hexameric DNL constructs (see, e.g., U.S. Pat. Nos. 7,550,143;7,521,056; 7,534,866; 7,527,787 and 7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNLconstruct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et at, Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Pre-Targeting

Bispecific or multispecific antibodies may be utilized in pre-targetingtechniques. Pre-targeting is a multistep process originally developed toresolve the slow blood clearance of directly targeting antibodies, whichcontributes to undesirable toxicity to normal tissues such as bonemarrow. With pre-targeting, a radionuclide or other diagnostic ortherapeutic agent is attached to a small delivery molecule (targetableconstruct) that is cleared within minutes from the blood. Apre-targeting bispecific or multispecific antibody, which has bindingsites for the targetable construct as well as a target antigen, isadministered first, free antibody is allowed to clear from circulationand then the targetable construct is administered.

Pre-targeting methods are disclosed, for example, in Goodwin et al.,U.S. Pat. No. 4,863,713; Goodwin et al., J. Nucl. Med. 29:226, 1988;Hnatowich et al., J. Nucl. Med. 28:1294, 1987; Oehr et al., J. Nucl.Med. 29:728, 1988; Klibanov et al., J. Nucl. Med. 29:1951, 1988;Sinitsyn et al., J. Nucl. Med. 30:66, 1989; Kalofonos et al., J. Nucl.Med. 31:1791, 1990; Schechter et al., Int. J. Cancer 48:167, 1991;Paganelli et al., Cancer Res. 51:5960, 1991; Paganelli et al., Nucl.Med. Commun. 12:211, 1991; U.S. Pat. No. 5,256,395; Stickney et al.,Cancer Res. 51:6650, 1991; Yuan et al., Cancer Res. 51:3119, 1991; U.S.Pat. Nos. 6,077,499; 7,011,812; 7,300,644; 7,074,405; 6,962,702;7,387,772; 7,052,872; 7,138,103; 6,090,381; 6,472,511; 6,962,702; and6,962,702, each incorporated herein by reference.

A pre-targeting method of treating or diagnosing a disease or disorderin a subject may be provided by: (1) administering to the subject abispecific antibody or antibody fragment; (2) optionally administeringto the subject a clearing composition, and allowing the composition toclear the antibody from circulation; and (3) administering to thesubject the targetable construct, containing one or more chelated orchemically bound therapeutic or diagnostic agents.

Immunoconjugates

Any of the antibodies, antibody fragments or antibody fusion proteinsdescribed herein may be conjugated to a chelating moiety or othercarrier molecule to form an immunoconjugate. Methods for covalentconjugation of chelating moieties and other functional groups are knownin the art and any such known method may be utilized.

For example, a chelating moiety or carrier can be attached at the hingeregion of a reduced antibody component via disulfide bond formation.Alternatively, such agents can be attached using a heterobifunctionalcross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP).Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for suchconjugation are well-known in the art. See, for example, Wong, CHEMISTRYOF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis etal., “Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995).

Alternatively, the chelating moiety or carrier can be conjugated via acarbohydrate moiety in the Fc region of the antibody. Methods forconjugating peptides to antibody components via an antibody carbohydratemoiety are well-known to those of skill in the art. See, for example,Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al., Int. J. Cancer46: 1101 (1990); and Shih et al., U.S. Pat. No. 5,057,313, the Examplessection of which is incorporated herein by reference. The general methodinvolves reacting an antibody component having an oxidized carbohydrateportion with a carrier polymer that has at least one free aminefunction. This reaction results in an initial Schiff base (imine)linkage, which can be stabilized by reduction to a secondary amine toform the final conjugate.

The Fc region may be absent if the antibody used as the antibodycomponent of the immunoconjugate is an antibody fragment. However, it ispossible to introduce a carbohydrate moiety into the light chainvariable region of a full length antibody or antibody fragment. See, forexample, Leung et al., J. Immunol. 154: 5919 (1995); U.S. Pat. Nos.5,443,953 and 6,254,868, the Examples section of which is incorporatedherein by reference. The engineered carbohydrate moiety is used toattach the functional group to the antibody fragment.

Other methods of conjugation of chelating agents to proteins are wellknown in the art (see, e.g., U.S. Pat. No. 7,563,433, the Examplessection of which is incorporated herein by reference). Chelates may bedirectly linked to antibodies or peptides, for example as disclosed inU.S. Pat. No. 4,824,659, incorporated herein in its entirety byreference.

Click Chemistry

In various embodiments, immunoconjugates may be prepared using the clickchemistry technology. The click chemistry approach was originallyconceived as a method to rapidly generate complex substances by joiningsmall subunits together in a modular fashion. (See, e.g., Kolb et al.,2004, Angew Chem Int Ed 40:3004-31; Evans, 2007, Aust J Chem 60:384-95.)Various forms of click chemistry reaction are known in the art, such asthe Huisgen 1,3-dipolar cycloaddition copper catalyzed reaction (Tornoeet al., 2002, J Organic Chem 67:3057-64), which is often referred to asthe “click reaction.” Other alternatives include cycloaddition reactionssuch as the Diels-Alder, nucleophilic substitution reactions (especiallyto small strained rings like epoxy and aziridine compounds), carbonylchemistry formation of urea compounds and reactions involvingcarbon-carbon double bonds, such as alkynes in thiol-yne reactions.

The azide alkyne Huisgen cycloaddition reaction uses a copper catalystin the presence of a reducing agent to catalyze the reaction of aterminal alkyne group attached to a first molecule. In the presence of asecond molecule comprising an azide moiety, the azide reacts with theactivated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The coppercatalyzed reaction occurs at room temperature and is sufficientlyspecific that purification of the reaction product is often notrequired. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe etal., 2002, J Org Chem 67:3057.) The azide and alkyne functional groupsare largely inert towards biomolecules in aqueous medium, allowing thereaction to occur in complex solutions. The triazole formed ischemically stable and is not subject to enzymatic cleavage, making theclick chemistry product highly stable in biological systems. However,the copper catalyst is toxic to living cells, precluding biologicalapplications.

A copper-free click reaction has been proposed for covalent modificationof biomolecules in living systems. (See, e.g., Agard et al., 2004, J AmChem Soc 126:15046-47.) The copper-free reaction uses ring strain inplace of the copper catalyst to promote a [3+2] azide-alkynecycloaddition reaction (Id.) For example, cyclooctyne is a 8-carbon ringstructure comprising an internal alkyne bond. The closed ring structureinduces a substantial bond angle deformation of the acetylene, which ishighly reactive with azide groups to form a triazole. Thus, cyclooctynederivatives may be used for copper-free click reactions, without thetoxic copper catalyst (Id.)

Another type of copper-free click reaction was reported by Ning et al.(2010, Angew Chem Int Ed 49:3065-68), involving strain-promotedalkyne-nitrone cycloaddition. To address the slow rate of the originalcyclooctyne reaction, electron-withdrawing groups are attached adjacentto the triple bond (Id.) Examples of such substituted cyclooctynesinclude difluorinated cyclooctynes, 4-dibenzocyclooctynol andazacyclooctyne (Id.) An alternative copper-free reaction involvedstrain-promoted alkyne-nitrone cycloaddition to give N-alkylatedisoxazolines (Id.) The reaction was reported to have exceptionally fastreaction kinetics and was used in a one-pot three-step protocol forsite-specific modification of peptides and proteins (Id.) Nitrones wereprepared by the condensation of appropriate aldehydes withN-methylhydroxylamine and the cycloaddition reaction took place in amixture of acetonitrile and water (Id.) However, an attempt to use thereaction with nitrone-labeled monosaccharide derivatives and metaboliclabeling in Jurkat cells was unsuccessful (Id.)

In some cases, activated groups for click chemistry reactions may beincorporated into biomolecules using the endogenous synthetic pathwaysof cells. For example, Agard et al. (2004, J Am Chem Soc 126:15046-47)demonstrated that a recombinant glycoprotein expressed in CHO cells inthe presence of peracetylated N-azidoacetylmannosamine resulted in theincorporation of the corresponding N-azidoacetyl sialic acid in thecarbohydrates of the glycoprotein. The azido-derivatized glycoproteinreacted specifically with a biotinylated cyclooctyne to form abiotinylated glycoprotein, while control glycoprotein without the azidomoiety remained unlabeled (Id.) Laughlin et al. (2008, Science320:664-667) used a similar technique to metabolically labelcell-surface glycans in zebrafish embryos incubated with peracetylatedN-azidoacetylgalactosamine. The azido-derivatized glycans reacted withdifluorinated cyclooctyne (DIFO) reagents to allow visualization ofglycans in vivo.

The Diels-Alder reaction has also been used for in vivo labeling ofmolecules. Rossin et al. (2010, Angew Chem Int Ed 49:3375-78) reported a52% yield in vivo between a tumor-localized anti-TAG72 (CC49) antibodycarrying a trans-cyclooctene (TCO) reactive moiety and an ¹¹¹In-labeledtetrazine DOTA derivative. The TCO-labeled CC49 antibody wasadministered to mice bearing colon cancer xenografts, followed 1 daylater by injection of ¹¹¹In-labeled tetrazine probe (Id.) The reactionof radiolabeled probe with tumor localized antibody resulted inpronounced radioactivity localized in the tumor, as demonstrated bySPECT imaging of live mice three hours after injection of radiolabeledprobe, with a tumor-to-muscle ratio of 13:1 (Id.) The results confirmedthe in vivo chemical reaction of the TCO and tetrazine-labeledmolecules.

Antibody labeling techniques using biological incorporation of labelingmoieties are further disclosed in U.S. Pat. No. 6,953,675 (the Examplessection of which is incorporated herein by reference). Such “landscaped”antibodies were prepared to have reactive ketone groups on glycosylatedsites. The method involved expressing cells transfected with anexpression vector encoding an antibody with one or more N-glycosylationsites in the CH1 or Vκ domain in culture medium comprising a ketonederivative of a saccharide or saccharide precursor. Ketone-derivatizedsaccharides or precursors included N-levulinoyl mannosamine andN-levulinoyl fucose. The landscaped antibodies were subsequently reactedwith agents comprising a ketone-reactive moiety, such as hydrazide,hydrazine, hydroxylamino or thiosemicarbazide groups, to form a labeledtargeting molecule. Exemplary agents attached to the landscapedantibodies included chelating agents like DTPA, large drug moleculessuch as doxorubicin-dextran, and acyl-hydrazide containing peptides.However, the landscaping technique is not limited to producingantibodies comprising ketone moieties, but may be used instead tointroduce a click chemistry reactive group, such as a nitrone, an azideor a cyclooctyne, onto an antibody or other biological molecule.

Modifications of click chemistry reactions are suitable for use in vitroor in vivo. Reactive targeting molecule may be formed either by eitherchemical conjugation or by biological incorporation. The targetingmolecule, such as an antibody or antibody fragment, may be activatedwith an azido moiety, a substituted cyclooctyne or alkyne group, or anitrone moiety. Where the targeting molecule comprises an azido ornitrone group, the corresponding targetable construct will comprise asubstituted cyclooctyne or alkyne group, and vice versa. Such activatedmolecules may be made by metabolic incorporation in living cells, asdiscussed above. Alternatively, methods of chemical conjugation of suchmoieties to biomolecules are well known in the art, and any such knownmethod may be utilized. The disclosed techniques may be used incombination with the ¹⁸F or ¹⁹F labeling methods described below for PETor NMR imaging, or alternatively may be utilized for delivery of anytherapeutic and/or diagnostic agent that may be conjugated to a suitableactivated targetable construct and/or targeting molecule.

Affibodies

Affibodies are small proteins that function as antibody mimetics and areof use in binding target molecules. Affibodies were developed bycombinatorial engineering on an alpha helical protein scaffold (Nord etal., 1995, Protein Eng 8:601-8; Nord et al., 1997, Nat Biotechnol15:772-77). The affibody design is based on a three helix bundlestructure comprising the IgG binding domain of protein A (Nord et al.,1995; 1997). Affibodies with a wide range of binding affinities may beproduced by randomization of thirteen amino acids involved in the Fcbinding activity of the bacterial protein A (Nord et al., 1995; 1997).After randomization, the PCR amplified library was cloned into aphagemid vector for screening by phage display of the mutant proteins.

A ¹⁷⁷Lu-labeled affibody specific for HER2/neu has been demonstrated totarget HER2-expressing xenografts in vivo (Tolmachev et al., 2007,Cancer Res 67:2773-82). Although renal toxicity due to accumulation ofthe low molecular weight radiolabeled compound was initially a problem,reversible binding to albumin reduced renal accumulation, enablingradionuclide-based therapy with labeled affibody (Id.)

The feasibility of using radiolabeled affibodies for in vivo tumorimaging has been recently demonstrated (Tolmachev et al., 2011,Bioconjugate Chem 22:894-902). A maleimide-derivatized NOTA wasconjugated to the anti-HER2 affibody and radiolabeled with ¹¹¹In (Id.)Administration to mice bearing the HER2-expressing DU-145 xenograft,followed by gamma camera imaging, allowed visualization of the xenograft(Id.)

The skilled artisan will realize that affibodies may be used astargeting molecules in the practice of the claimed methods andcompositions. Labeling with metal-conjugated ¹⁸F may be performed asdescribed in the Examples below. Affibodies are commercially availablefrom Affibody AB (Solna, Sweden).

Phage Display Peptides

In some alternative embodiments, binding peptides may be produced byphage display methods that are well known in the art. For example,peptides that bind to any of a variety of disease-associated antigensmay be identified by phage display panning against an appropriate targetantigen, cell, tissue or pathogen and selecting for phage with highbinding affinity.

Various methods of phage display and techniques for producing diversepopulations of peptides are well known in the art. For example, U.S.Pat. Nos. 5,223,409; 5,622,699 and 6,068,829, each of which isincorporated herein by reference, disclose methods for preparing a phagelibrary. The phage display technique involves genetically manipulatingbacteriophage so that small peptides can be expressed on their surface(Smith and Scott, 1985, Science 228:1315-1317; Smith and Scott, 1993,Meth. Enzymol. 21:228-257).

The past decade has seen considerable progress in the construction ofphage-displayed peptide libraries and in the development of screeningmethods in which the libraries are used to isolate peptide ligands. Forexample, the use of peptide libraries has made it possible tocharacterize interacting sites and receptor-ligand binding motifs withinmany proteins, such as antibodies involved in inflammatory reactions orintegrins that mediate cellular adherence. This method has also beenused to identify novel peptide ligands that may serve as leads to thedevelopment of peptidomimetic drugs or imaging agents (Arap et al.,1998a, Science 279:377-380). In addition to peptides, larger proteindomains such as single-chain antibodies may also be displayed on thesurface of phage particles (Arap et al., 1998a).

Targeting amino acid sequences selective for a given target molecule maybe isolated by panning (Pasqualini and Ruoslahti, 1996, Nature380:364-366; Pasqualini, 1999, The Quart. J. Nucl. Med. 43:159-162). Inbrief, a library of phage containing putative targeting peptides isadministered to target molecules and samples containing bound phage arecollected. Target molecules may, for example, be attached to the bottomof microtiter wells in a 96-well plate. Phage that bind to a target maybe eluted and then amplified by growing them in host bacteria.

In certain embodiments, the phage may be propagated in host bacteriabetween rounds of panning. Rather than being lysed by the phage, thebacteria may instead secrete multiple copies of phage that display aparticular insert. If desired, the amplified phage may be exposed to thetarget molecule again and collected for additional rounds of panning.Multiple rounds of panning may be performed until a population ofselective or specific binders is obtained. The amino acid sequence ofthe peptides may be determined by sequencing the DNA corresponding tothe targeting peptide insert in the phage genome. The identifiedtargeting peptide may then be produced as a synthetic peptide bystandard protein chemistry techniques (Arap et al., 1998a, Smith et al.,1985).

Aptamers

In certain embodiments, a targeting molecule may comprise an aptamer.Methods of constructing and determining the binding characteristics ofaptamers are well known in the art. For example, such techniques aredescribed in U.S. Pat. Nos. 5,582,981, 5,595,877 and 5,637,459, eachincorporated herein by reference.

Aptamers may be prepared by any known method, including synthetic,recombinant, and purification methods, and may be used alone or incombination with other ligands specific for the same target. In general,a minimum of approximately 3 nucleotides, preferably at least 5nucleotides, are necessary to effect specific binding. Aptamers ofsequences shorter than 10 bases may be feasible, although aptamers of10, 20, 30 or 40 nucleotides may be preferred.

Aptamers need to contain the sequence that confers binding specificity,but may be extended with flanking regions and otherwise derivatized. Inpreferred embodiments, the binding sequences of aptamers may be flankedby primer-binding sequences, facilitating the amplification of theaptamers by PCR or other amplification techniques. In a furtherembodiment, the flanking sequence may comprise a specific sequence thatpreferentially recognizes or binds a moiety to enhance theimmobilization of the aptamer to a substrate.

Aptamers may be isolated, sequenced, and/or amplified or synthesized asconventional DNA or RNA molecules. Alternatively, aptamers of interestmay comprise modified oligomers. Any of the hydroxyl groups ordinarilypresent in aptamers may be replaced by phosphonate groups, phosphategroups, protected by a standard protecting group, or activated toprepare additional linkages to other nucleotides, or may be conjugatedto solid supports. One or more phosphodiester linkages may be replacedby alternative linking groups, such as P(O)O replaced by P(O)S,P(O)NR.sub.2, P(O)R, P(O)OR′, CO, or CNR.sub.2, wherein R is H or alkyl(1-20C) and R′ is alkyl (1-20C); in addition, this group may be attachedto adjacent nucleotides through O or S. Not all linkages in an oligomerneed to be identical.

Methods for preparation and screening of aptamers that bind toparticular targets of interest are well known, for example U.S. Pat. No.5,475,096 and U.S. Pat. No. 5,270,163, each incorporated by reference.The technique generally involves selection from a mixture of candidateaptamers and step-wise iterations of binding, separation of bound fromunbound aptamers and amplification. Because only a small number ofsequences (possibly only one molecule of aptamer) corresponding to thehighest affinity aptamers exist in the mixture, it is generallydesirable to set the partitioning criteria so that a significant amountof aptamers in the mixture (approximately 5-50%) is retained duringseparation. Each cycle results in an enrichment of aptamers with highaffinity for the target. Repetition for between three to six selectionand amplification cycles may be used to generate aptamers that bind withhigh affinity and specificity to the target.

Avimers

In certain embodiments, the targeting molecules may comprise one or moreavimer sequences. Avimers are a class of binding proteins somewhatsimilar to antibodies in their affinities and specificities for varioustarget molecules. They were developed from human extracellular receptordomains by in vitro exon shuffling and phage display. (Silverman et al.,2005, Nat. Biotechnol. 23:1493-94; Silverman et al., 2006, Nat.Biotechnol. 24:220.) The resulting multidomain proteins may comprisemultiple independent binding domains, that may exhibit improved affinity(in some cases sub-nanomolar) and specificity compared withsingle-epitope binding proteins. (Id.) Additional details concerningmethods of construction and use of avimers are disclosed, for example,in U.S. Patent Application Publication Nos. 20040175756, 20050048512,20050053973, 20050089932 and 20050221384, the Examples section of eachof which is incorporated herein by reference.

Methods of Administration

In various embodiments, bispecific antibodies and targetable constructsmay be used for imaging normal or diseased tissue and organs (see, e.g.U.S. Pat. Nos. 6,126,916; 6,077,499; 6,010,680; 5,776,095; 5,776,094;5,776,093; 5,772,981; 5,753,206; 5,746,996; 5,697,902; 5,328,679;5,128,119; 5,101,827; and 4,735,210, each incorporated herein byreference in its Examples section).

The administration of a bispecific antibody (bsAb) and an ¹⁸F-labeledtargetable construct may be conducted by administering the bsAb antibodyat some time prior to administration of the targetable construct. Thedoses and timing of the reagents can be readily devised by a skilledartisan, and are dependent on the specific nature of the reagentsemployed. If a bsAb-F(ab′)₂ derivative is given first, then a waitingtime of 24-72 hr (alternatively 48-96 hours) before administration ofthe targetable construct would be appropriate. If an IgG-Fab′ bsAbconjugate is the primary targeting vector, then a longer waiting periodbefore administration of the targetable construct would be indicated, inthe range of 3-10 days. After sufficient time has passed for the bsAb totarget to the diseased tissue, the ¹⁸F-labeled targetable construct isadministered. Subsequent to administration of the targetable construct,imaging can be performed.

Certain embodiments concern the use of multivalent target bindingproteins which have at least three different target binding sites asdescribed in patent application Ser. No. 60/220,782. Multivalent targetbinding proteins have been made by cross-linking several Fab-likefragments via chemical linkers. See U.S. Pat. Nos. 5,262,524; 5,091,542and Landsdorp et al. Euro. J. Immunol. 16: 679-83 (1986). Multivalenttarget binding proteins also have been made by covalently linkingseveral single chain Fv molecules (scFv) to form a single polypeptide.See U.S. Pat. No. 5,892,020. A multivalent target binding protein whichis basically an aggregate of scFv molecules has been disclosed in U.S.Pat. Nos. 6,025,165 and 5,837,242. A trivalent target binding proteincomprising three scFv molecules has been described in Krott et al.Protein Engineering 10(4): 423-433 (1997).

Alternatively, a technique known as “dock-and-lock” (DNL), described inmore detail below, has been demonstrated for the simple and reproducibleconstruction of a variety of multivalent complexes, including complexescomprising two or more different antibodies or antibody fragments. (See,e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787 and7,666,400, the Examples section of each of which is incorporated hereinby reference) Such constructs are also of use for the practice of theclaimed methods and compositions described herein.

A clearing agent may be used which is given between doses of thebispecific antibody (bsAb) and the targetable construct. A clearingagent of novel mechanistic action may be used, namely a glycosylatedanti-idiotypic Fab′ fragment targeted against the disease targetingarm(s) of the bsAb. In one example, anti-CEA (MN-14 Ab)×anti-peptidebsAb is given and allowed to accrete in disease targets to its maximumextent. To clear residual bsAb from circulation, an anti-idiotypic Ab toMN-14, termed WI2, is given, preferably as a glycosylated Fab′ fragment.The clearing agent binds to the bsAb in a monovalent manner, while itsappended glycosyl residues direct the entire complex to the liver, whererapid metabolism takes place. Then the ¹⁸F-labeled targetable constructis given to the subject. The WI2 Ab to the MN-14 arm of the bsAb has ahigh affinity and the clearance mechanism differs from other disclosedmechanisms (see Goodwin et al., ibid), as it does not involvecross-linking, because the WI2-Fab′ is a monovalent moiety. However,alternative methods and compositions for clearing agents are known andany such known clearing agents may be used.

Formulation and Administration

The ¹⁸F-labeled molecules may be formulated to obtain compositions thatinclude one or more pharmaceutically suitable excipients, one or moreadditional ingredients, or some combination of these. These can beaccomplished by known methods to prepare pharmaceutically usefuldosages, whereby the active ingredients (i.e., the ¹⁸F-labeledmolecules) are combined in a mixture with one or more pharmaceuticallysuitable excipients. Sterile phosphate-buffered saline is one example ofa pharmaceutically suitable excipient. Other suitable excipients arewell known to those in the art. See, e.g., Ansel et al., PHARMACEUTICALDOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger1990), and Gennaro (ed.), REMINGTON'S PHARMACEUTICAL SCIENCES, 18thEdition (Mack Publishing Company 1990), and revised editions thereof.

The preferred route for administration of the compositions describedherein is parenteral injection. Injection may be intravenous,intraarterial, intralymphatic, intrathecal, subcutaneous orintracavitary (i.e., parenterally). In parenteral administration, thecompositions will be formulated in a unit dosage injectable form such asa solution, suspension or emulsion, in association with apharmaceutically acceptable excipient. Such excipients are inherentlynontoxic and nontherapeutic. Examples of such excipients are saline,Ringer's solution, dextrose solution and Hank's solution. Nonaqueousexcipients such as fixed oils and ethyl oleate may also be used. Apreferred excipient is 5% dextrose in saline. The excipient may containminor amounts of additives such as substances that enhance isotonicityand chemical stability, including buffers and preservatives. Othermethods of administration, including oral administration, are alsocontemplated.

Formulated compositions comprising ¹⁸F-labeled molecules can be used forintravenous administration via, for example, bolus injection orcontinuous infusion. Compositions for injection can be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. Compositions can also take such forms assuspensions, solutions or emulsions in oily or aqueous vehicles, and cancontain formulatory agents such as suspending, stabilizing and/ordispersing agents. Alternatively, the compositions can be in powder formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

The compositions may be administered in solution. The pH of the solutionshould be in the range of pH 5 to 9.5, preferably pH 6.5 to 7.5. Theformulation thereof should be in a solution having a suitablepharmaceutically acceptable buffer such as phosphate,TRIS(hydroxymethyl)aminomethane-HCl or citrate and the like. In certainpreferred embodiments, the buffer is potassium biphthalate (KHP), whichmay act as a transfer ligand to facilitate ¹⁸F-labeling. Bufferconcentrations should be in the range of 1 to 100 mM. The formulatedsolution may also contain a salt, such as sodium chloride or potassiumchloride in a concentration of 50 to 150 mM. An effective amount of astabilizing agent such as glycerol, albumin, a globulin, a detergent, agelatin, a protamine or a salt of protamine may also be included. Thecompositions may be administered to a mammal subcutaneously,intravenously, intramuscularly or by other parenteral routes. Moreover,the administration may be by continuous infusion or by single ormultiple boluses.

Where bispecific antibodies are administered, for example in apretargeting technique, the dosage of an administered antibody forhumans will vary depending upon such factors as the patient's age,weight, height, sex, general medical condition and previous medicalhistory. Typically, for imaging purposes it is desirable to provide therecipient with a dosage of bispecific antibody that is in the range offrom about 1 mg to 200 mg as a single intravenous infusion, although alower or higher dosage also may be administered as circumstancesdictate. Typically, it is desirable to provide the recipient with adosage that is in the range of from about 10 mg per square meter of bodysurface area or 17 to 18 mg of the antibody for the typical adult,although a lower or higher dosage also may be administered ascircumstances dictate. Examples of dosages of bispecific antibodies thatmay be administered to a human subject for imaging purposes are 1 to 200mg, more preferably 1 to 70 mg, most preferably 1 to 20 mg, althoughhigher or lower doses may be used.

In general, the dosage of ¹⁸F label to administer will vary dependingupon such factors as the patient's age, weight, height, sex, generalmedical condition and previous medical history. Preferably, a saturatingdose of the ¹⁸F-labeled molecules is administered to a patient. Foradministration of ¹⁸F-labeled molecules, the dosage may be measured bymillicuries. A typical range for ¹⁸F imaging studies would be five to 10mCi.

Administration of Peptides

Various embodiments of the claimed methods and/or compositions mayconcern one or more ¹⁸F-labeled peptides to be administered to asubject. Administration may occur by any route known in the art,including but not limited to oral, nasal, buccal, inhalational, rectal,vaginal, topical, orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal, intraarterial, intrathecal or intravenous injection.Where, for example, ¹⁸F-labeled peptides are administered in apretargeting protocol, the peptides would preferably be administeredi.v.

In certain embodiments, the standard peptide bond linkage may bereplaced by one or more alternative linking groups, such as CH₂—NH,CH₂—S, CH₂—CH₂, CH═CH, CO—CH₂, CHOH—CH₂ and the like. Methods forpreparing peptide mimetics are well known (for example, Hruby, 1982,Life Sci 31:189-99; Holladay et al., 1983, Tetrahedron Lett. 24:4401-04;Jennings-White et al., 1982, Tetrahedron Lett. 23:2533; Almquiest etal., 1980, J. Med. Chem. 23:1392-98; Hudson et al., 1979, Int. J. Pept.Res. 14:177-185; Spatola et al., 1986, Life Sci 38:1243-49; U.S. Pat.Nos. 5,169,862; 5,539,085; 5,576,423, 5,051,448, 5,559,103.) Peptidemimetics may exhibit enhanced stability and/or absorption in vivocompared to their peptide analogs.

Peptide stabilization may also occur by substitution of D-amino acidsfor naturally occurring L-amino acids, particularly at locations whereendopeptidases are known to act. Endopeptidase binding and cleavagesequences are known in the art and methods for making and using peptidesincorporating D-amino acids have been described (e.g., U.S. PatentApplication Publication No. 20050025709, McBride et al., filed Jun. 14,2004, the Examples section of which is incorporated herein byreference).

Imaging Using Labeled Molecules

Methods of imaging using labeled molecules are well known in the art,and any such known methods may be used with the ¹⁸F-labeled moleculesdisclosed herein. See, e.g., U.S. Pat. Nos. 6,241,964; 6,358,489;6,953,567 and published U.S. Patent Application Publ. Nos. 20050003403;20040018557; 20060140936, the Examples section of each incorporatedherein by reference. See also, Page et al., Nuclear Medicine AndBiology, 21:911-919, 1994; Choi et al., Cancer Research 55:5323-5329,1995; Zalutsky et al., J. Nuclear Med., 33:575-582, 1992; Woessner et.al. Magn. Reson. Med. 2005, 53: 790-99.

In certain embodiments, ¹⁸F-labeled molecules may be of use in imagingnormal or diseased tissue and organs, for example using the methodsdescribed in U.S. Pat. Nos. 6,126,916; 6,077,499; 6,010,680; 5,776,095;5,776,094; 5,776,093; 5,772,981; 5,753,206; 5,746,996; 5,697,902;5,328,679; 5,128,119; 5,101,827; and 4,735,210, each incorporated hereinby reference. Such imaging can be conducted by direct ¹⁸F labeling ofthe appropriate targeting molecules, or by a pretargeted imaging method,as described in Goldenberg et al. (2007, Update Cancer Ther. 2:19-31);Sharkey et al. (2008, Radiology 246:497-507); Goldenberg et al. (2008,J. Nucl. Med. 49:158-63); Sharkey et al. (2007, Clin. Cancer Res.13:5777s-5585s); McBride et al. (2006, J. Nucl. Med. 47:1678-88);Goldenberg et al. (2006, J. Clin. Oncol. 24:823-85), see also U.S.Patent Publication Nos. 20050002945, 20040018557, 20030148409 and20050014207, each incorporated herein by reference.

Methods of diagnostic imaging with labeled peptides or MAbs arewell-known. For example, in the technique of immunoscintigraphy, ligandsor antibodies are labeled with a gamma-emitting radioisotope andintroduced into a patient. A gamma camera is used to detect the locationand distribution of gamma-emitting radioisotopes. See, for example,Srivastava (ed.), RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING ANDTHERAPY (Plenum Press 1988), Chase, “Medical Applications ofRadioisotopes,” in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition,Gennaro et al. (eds.), pp. 624-652 (Mack Publishing Co., 1990), andBrown, “Clinical Use of Monoclonal Antibodies,” in BIOTECHNOLOGY ANDPHARMACY 227-49, Pezzuto et al. (eds.) (Chapman & Hall 1993). Alsopreferred is the use of positron-emitting radionuclides (PET isotopes),such as with an energy of 511 keV, such as ¹⁸F, ⁶⁸Ga, ⁶⁴Cu, and ¹²⁴I.Such radionuclides may be imaged by well-known PET scanning techniques.

In preferred embodiments, the ¹⁸F-labeled peptides, proteins and/orantibodies are of use for imaging of cancer. Examples of cancersinclude, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,and leukemia or lymphoid malignancies. More particular examples of suchcancers are noted below and include: squamous cell cancer (e.g.epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer, adenocarcinoma of the lung andsquamous carcinoma of the lung, cancer of the peritoneum, hepatocellularcancer, gastric or stomach cancer including gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectalcancer, colorectal cancer, endometrial cancer or uterine carcinoma,salivary gland carcinoma, kidney or renal cancer, prostate cancer,vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penilecarcinoma, as well as head and neck cancer. The term “cancer” includesprimary malignant cells or tumors (e.g., those whose cells have notmigrated to sites in the subject's body other than the site of theoriginal malignancy or tumor) and secondary malignant cells or tumors(e.g., those arising from metastasis, the migration of malignant cellsor tumor cells to secondary sites that are different from the site ofthe original tumor).

Other examples of cancers or malignancies include, but are not limitedto: Acute Childhood Lymphoblastic Leukemia, Acute LymphoblasticLeukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult(Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult AcuteMyeloid Leukemia, Adult Hodgkin's Disease, Adult Hodgkin's Lymphoma,Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult PrimaryLiver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma,AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer,Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, BreastCancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System(Primary) Lymphoma, Central Nervous System Lymphoma, CerebellarAstrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary)Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood AcuteLymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, ChildhoodBrain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood CerebralAstrocytoma, Childhood Extracranial Germ Cell Tumors, ChildhoodHodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamicand Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, ChildhoodMedulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal andSupratentorial Primitive Neuroectodermal Tumors, Childhood Primary LiverCancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma,Childhood Visual Pathway and Hypothalamic Glioma, Chronic LymphocyticLeukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T-CellLymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer,Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma andRelated Tumors, Exocrine Pancreatic Cancer, Extracranial Germ CellTumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, EyeCancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer,Gastric Cancer, Gastrointestinal Carcinoid Tumor, GastrointestinalTumors, Germ Cell Tumors, Gestational Trophoblastic Tumor, Hairy CellLeukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin'sDisease, Hodgkin's Lymphoma, Hypergammaglobulinemia, HypopharyngealCancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma,Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, LaryngealCancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer,Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer,Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma,Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, MetastaticPrimary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, MultipleMyeloma, Multiple Myeloma/Plasma Cell Neoplasm, MyelodysplasticSyndrome, Myelogenous Leukemia, Myeloid Leukemia, MyeloproliferativeDisorders, Nasal Cavity and Paranasal Sinus Cancer, NasopharyngealCancer, Neuroblastoma, Non-Hodgkin's Lymphoma During Pregnancy,Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult PrimaryMetastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo-/MalignantFibrous Sarcoma, Osteosarcoma/Malignant Fibrous Histiocytoma,Osteosarcoma/Malignant Fibrous Histiocytoma of Bone, Ovarian EpithelialCancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor,Pancreatic Cancer, Paraproteinemias, Purpura, Parathyroid Cancer, PenileCancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/MultipleMyeloma, Primary Central Nervous System Lymphoma, Primary Liver Cancer,Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis andUreter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer,Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell LungCancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous NeckCancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal andPineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, ThyroidCancer, Transitional Cell Cancer of the Renal Pelvis and Ureter,Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors,Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer,Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma,Vulvar Cancer, Waldenstrom's Macroglobulinemia, Wilms' Tumor, and anyother hyperproliferative disease, besides neoplasia, located in an organsystem listed above.

The methods and compositions described and claimed herein may be used todetect or diagnose malignant or premalignant conditions. Such uses areindicated in conditions known or suspected of preceding progression toneoplasia or cancer, in particular, where non-neoplastic cell growthconsisting of hyperplasia, metaplasia, or most particularly, dysplasiahas occurred (for review of such abnormal growth conditions, see Robbinsand Angell, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia,pp. 68-79 (1976)).

Dysplasia is frequently a forerunner of cancer, and is found mainly inthe epithelia. It is the most disorderly form of non-neoplastic cellgrowth, involving a loss in individual cell uniformity and in thearchitectural orientation of cells. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation. Dysplasticdisorders which can be detected include, but are not limited to,anhidrotic ectodermal dysplasia, anterofacial dysplasia, asphyxiatingthoracic dysplasia, atriodigital dysplasia, bronchopulmonary dysplasia,cerebral dysplasia, cervical dysplasia, chondroectodermal dysplasia,cleidocranial dysplasia, congenital ectodermal dysplasia,craniodiaphysial dysplasia, craniocarpotarsal dysplasia,craniometaphysial dysplasia, dentin dysplasia, diaphysial dysplasia,ectodermal dysplasia, enamel dysplasia, encephalo-ophthalmic dysplasia,dysplasia epiphysialis hemimelia, dysplasia epiphysialis multiplex,dysplasia epiphysialis punctata, epithelial dysplasia,faciodigitogenital dysplasia, familial fibrous dysplasia of jaws,familial white folded dysplasia, fibromuscular dysplasia, fibrousdysplasia of bone, florid osseous dysplasia, hereditary renal-retinaldysplasia, hidrotic ectodermal dysplasia, hypohidrotic ectodermaldysplasia, lymphopenic thymic dysplasia, mammary dysplasia,mandibulofacial dysplasia, metaphysial dysplasia, Mondini dysplasia,monostotic fibrous dysplasia, mucoepithelial dysplasia, multipleepiphysial dysplasia, oculoauriculovertebral dysplasia,oculodentodigital dysplasia, oculovertebral dysplasia, odontogenicdysplasia, opthalmomandibulomelic dysplasia, periapical cementaldysplasia, polyostotic fibrous dysplasia, pseudoachondroplasticspondyloepiphysial dysplasia, retinal dysplasia, septo-optic dysplasia,spondyloepiphysial dysplasia, and ventriculoradial dysplasia.

Additional pre-neoplastic disorders which can be detected include, butare not limited to, benign dysproliferative disorders (e.g., benigntumors, fibrocystic conditions, tissue hypertrophy, intestinal polyps,colon polyps, and esophageal dysplasia), leukoplakia, keratoses, Bowen'sdisease, Farmer's Skin, solar cheilitis, and solar keratosis.

Additional hyperproliferative diseases, disorders, and/or conditionsinclude, but are not limited to, progression, and/or metastases ofmalignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,emangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

In a preferred embodiment, diseases that may be diagnosed, detected orimaged using the claimed compositions and methods include cardiovasculardiseases, such as fibrin clots, atherosclerosis, myocardial ischemia andinfarction. Antibodies to fibrin (e.g., scFv(59D8); T2G1s; MH1) areknown and in clinical trials as imaging agents for disclosing said clotsand pulmonary emboli, while anti-granulocyte antibodies, such as MN-3,MN-15, anti-NCA95, and anti-CD15 antibodies, can target myocardialinfarcts and myocardial ischemia. (See, e.g., U.S. Pat. Nos. 5,487,892;5,632,968; 6,294,173; 7,541,440, the Examples section of eachincorporated herein by reference) Anti-macrophage, anti-low-densitylipoprotein (LDL) and anti-CD74 (e.g., hLL1) antibodies can be used totarget atherosclerotic plaques. Abciximab (anti-glycoprotein IIb/IIIa)has been approved for adjuvant use for prevention of restenosis inpercutaneous coronary interventions and the treatment of unstable angina(Waldmann et al., 2000, Hematol 1:394-408). Anti-CD3 antibodies havebeen reported to reduce development and progression of atherosclerosis(Steffens et al., 2006, Circulation 114:1977-84). Treatment withblocking MIF antibody has been reported to induce regression ofestablished atherosclerotic lesions (Sanchez-Madrid and Sessa, 2010,Cardiovasc Res 86:171-73). Antibodies against oxidized LDL also induceda regression of established atherosclerosis in a mouse model (Ginsberg,2007, J Am Coll Cardiol 52:2319-21). Anti-ICAM-1 antibody was shown toreduce ischemic cell damage after cerebral artery occlusion in rats(Zhang et al., 1994, Neurology 44:1747-51). Commercially availablemonoclonal antibodies to leukocyte antigens are represented by: OKTanti-T-cell monoclonal antibodies (available from Ortho PharmaceuticalCompany) which bind to normal T-lymphocytes; the monoclonal antibodiesproduced by the hybridomas having the ATCC accession numbers HB44, HB55,HB12, HB78 and HB2; G7E11, W8E7, NKP15 and G022 (Becton Dickinson);NEN9.4 (New England Nuclear); and FMC11 (Sera Labs). A description ofantibodies against fibrin and platelet antigens is contained in Knight,Semin. Nucl. Med., 20:52-67 (1990).

In one embodiment, a pharmaceutical composition may be used to diagnosea subject having a metabolic disease, such amyloidosis, or aneurodegenerative disease, such as Alzheimer's disease, amyotrophiclateral sclerosis (ALS), Parkinson's disease, Huntington's disease,olivopontocerebellar atrophy, multiple system atrophy, progressivesupranuclear palsy, corticodentatonigral degeneration, progressivefamilial myoclonic epilepsy, strionigral degeneration, torsion dystonia,familial tremor, Gilles de la Tourette syndrome or Hallervorden-Spatzdisease. Bapineuzumab is in clinical trials for Alzheimer's diseasetherapy. Other antibodies proposed for Alzheimer's disease include Alz50 (Ksiezak-Reding et al., 1987, J Biol Chem 263:7943-47), gantenerumab,and solanezumab. Infliximab, an anti-TNF-α antibody, has been reportedto reduce amyloid plaques and improve cognition. Antibodies againstmutant SOD1, produced by hybridoma cell lines deposited with theInternational Depositary Authority of Canada (accession Nos.ADI-290806-01, ADI-290806-02, ADI-290806-03) have been proposed fortherapy of ALS, Parkinson's disease and Alzheimer's disease (see U.S.Patent Appl. Publ. No. 20090068194). Anti-CD3 antibodies have beenproposed for therapy of type 1 diabetes (Cernea et al., 2010, DiabetesMetab Rev 26:602-05). In addition, a pharmaceutical composition of thepresent invention may be used on a subject having animmune-dysregulatory disorder, such as graft-versus-host disease ororgan transplant rejection.

The exemplary conditions listed above that may be detected, diagnosedand/or imaged are not limiting. The skilled artisan will be aware thatantibodies, antibody fragments or targeting peptides are known for awide variety of conditions, such as autoimmune disease, cardiovasculardisease, neurodegenerative disease, metabolic disease, cancer,infectious disease and hyperproliferative disease. Any such conditionfor which an ¹⁸F-labeled molecule, such as a protein or peptide, may beprepared and utilized by the methods described herein, may be imaged,diagnosed and/or detected as described herein.

Kits

Various embodiments may concern kits containing components suitable forimaging, diagnosing and/or detecting diseased tissue in a patient usinglabeled compounds. Exemplary kits may contain an antibody, fragment orfusion protein, such as a bispecific antibody of use in pretargetingmethods as described herein. Other components may include a targetableconstruct for use with such bispecific antibodies. In preferredembodiments, the targetable construct is pre-conjugated to a chelatinggroup that may be used to attach an Al¹⁸F complex or a complex of ¹⁸Fwith a different metal. However, in alternative embodiments it iscontemplated that a targetable construct may be attached to one or moredifferent diagnostic agents, such as ⁶⁸Ga.

A device capable of delivering the kit components may be included. Onetype of device, for applications such as parenteral delivery, is asyringe that is used to inject the composition into the body of asubject. Inhalation devices may also be used for certain applications.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES Example 1 ¹⁸F-Labeling of Peptide IMP272

The first peptide that was prepared and ¹⁸F-labeled was IMP272:

(SEQ ID NO: 3) DTPA-Gln-Ala-Lys(HSG)-D-Tyr-Lys(HSG)-NH₂

Acetate buffer solution—Acetic acid, 1.509 g was diluted in ˜160 mLwater and the pH was adjusted by the addition of 1 M NaOH then dilutedto 250 mL to make a 0.1 M solution at pH 4.03.

Aluminum acetate buffer solution—A solution of aluminum was prepared bydissolving 0.1028 g of AlCl₃ hexahydrate in 42.6 mL DI water. A 4 mLaliquot of the aluminum solution was mixed with 16 mL of a 0.1 M NaOAcsolution at pH 4 to provide a 2 mM Al stock solution.

IMP272 acetate buffer solution—Peptide, 0.0011 g, 7.28×10⁻⁷ mol IMP272was dissolved in 364 μL of the 0.1 M pH 4 acetate buffer solution toobtain a 2 mM stock solution of the peptide.

¹⁸F-Labeling of IMP272—A 3 μL aliquot of the aluminum stock solution wasplaced in a REACTI-VIAL™ and mixed with 50 μL ¹⁸F (as received) and 3 μLof the IMP272 solution. The solution was heated in a heating block at110° C. for 15 min and analyzed by reverse phase HPLC. The HPLC trace(not shown) showed 93% free ¹⁸F and 7% bound to the peptide. Anadditional 10 μL of the IMP272 solution was added to the reaction and itwas heated again and analyzed by reverse phase HPLC (not shown). TheHPLC trace showed 8% ¹⁸F at the void volume and 92% of the activityattached to, the peptide. The remainder of the peptide solution wasincubated at room temperature with 150 μL PBS for ˜1 hr and thenexamined by reverse phase HPLC. The HPLC (not shown) showed 58% ¹⁸Funbound and 42% still attached to the peptide. The data indicate thatAl¹⁸F(DTPA) complex may be unstable when mixed with phosphate.

Example 2 IMP272 ¹⁸F-Labeling with Other Metals

A ˜3 μL aliquot of the metal stock solution (6×10⁻⁹ mol) was placed in apolypropylene cone vial and mixed with 75 μL ¹⁸F (as received),incubated at room temperature for ˜2 min and then mixed with 20 μL of a2 mM (4×10⁻⁸ mol) IMP272 solution in 0.1 M NaOAc pH 4 buffer. Thesolution was heated in a heating block at 100° C. for 15 min andanalyzed by reverse phase HPLC. IMP272 was labeled with indium (24%),gallium (36%), zirconium (15%), lutetium (37%) and yttrium (2%) (notshown). These results demonstrate that the ¹⁸F metal labeling techniqueis not limited to an aluminum ligand, but can also utilize other metalsas well. With different metal ligands, different chelating moieties maybe utilized to optimize binding of an ¹⁸F-metal conjugate.

Example 3 Production and Use of a Serum-Stable ¹⁸F-Labeled PeptideIMP449

(SEQ ID NO: 4) NOTA-benzyl-ITC-D-Ala-D-Lys(HSG)-D-Tyr-D- Lys(HSG)-NH₂

The peptide, IMP448 D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (SEQ ID NO:5)was made on Sieber Amide resin by adding the following amino acids tothe resin in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Alocwas cleaved, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, theAloc was cleaved, Fmoc-D-Ala-OH with final Fmoc cleavage to make thedesired peptide. The peptide was then cleaved from the resin andpurified by HPLC to produce IMP448, which was then coupled to ITC-benzylNOTA.

IMP448 (0.0757 g, 7.5×10⁻⁵ mol) was mixed with 0.0509 g (9.09×10⁻⁵ mol)ITC benzyl NOTA and dissolved in 1 mL water. Potassium carbonateanhydrous (0.2171 g) was then slowly added to the stirred peptide/NOTAsolution. The reaction solution was pH 10.6 after the addition of allthe carbonate. The reaction was allowed to stir at room temperatureovernight. The reaction was carefully quenched with 1 M HCl after 14 hrand purified by HPLC to obtain 48 mg of IMP449.

¹⁸F-Labeling of IMP449

IMP449 (0.002 g, 1.37×10⁻⁶ mol) was dissolved in 686 μL (2 mM peptidesolution) 0.1 M NaOAc pH 4.02. Three microliters of a 2 mM solution ofAl in a pH 4 acetate buffer was mixed with 15 μL, 1.3 mCi of ¹⁸F. Thesolution was then mixed with 20 μL of the 2 mM IMP449 solution andheated at 105° C. for 15 min. Reverse Phase HPLC analysis showed 35%(t_(R)˜10 min) of the activity was attached to the peptide and 65% ofthe activity was eluted at the void volume of the column (3.1 min, notshown) indicating that the majority of activity was not associated withthe peptide. The crude labeled mixture (5 μL) was mixed with pooledhuman serum and incubated at 37° C. An aliquot was removed after 15 minand analyzed by HPLC. The HPLC showed 9.8% of the activity was stillattached to the peptide (down from 35%). Another aliquot was removedafter 1 hr and analyzed by HPLC. The HPLC showed 7.6% of the activitywas still attached to the peptide (down from 35%), which was essentiallythe same as the 15 min trace (data not shown).

High Dose ¹⁸F-Labeling of IMP449

Further studies with purified IMP449 demonstrated that the ¹⁸F-labeledpeptide was highly stable (91%, not shown) in human serum at 37° C. forat least one hour and was partially stable (76%, not shown) in humanserum at 37° C. for at least four hours. Additional studies wereperformed in which the IMP449 was prepared in the presence of ascorbicacid as a stabilizing agent. In those studies (not shown), the¹⁸F-metal-peptide complex showed no detectable decomposition in serumafter 4 hr at 37° C. The mouse urine 30 min after injection of¹⁸F-labeled peptide was found to contain ¹⁸F bound to the peptide (notshown). These results demonstrate that the ¹⁸F-labeled peptidesdisclosed herein exhibit sufficient stability under approximated in vivoconditions to be used for ¹⁸F imaging studies.

Since IMP449 peptide contains a thiourea linkage, which is sensitive toradiolysis, several products are observed by RP-HPLC. However, whenascorbic acid is added to the reaction mixture, the side productsgenerated are markedly reduced.

Example 4 Preparation of DNL Constructs for ¹⁸F Imaging by Pretargeting

The DNL technique may be used to make dimers, trimers, tetramers,hexamers, etc. comprising virtually any antibodies or fragments thereofor other effector moieties. For certain preferred embodiments, IgGantibodies, Fab fragments or other proteins or peptides may be producedas fusion proteins containing either a DDD (dimerization and dockingdomain) or AD (anchoring domain) sequence. Bispecific antibodies may beformed by combining a Fab-DDD fusion protein of a first antibody with aFab-AD fusion protein of a second antibody. Alternatively, constructsmay be made that combine IgG-AD fusion proteins with Fab-DDD fusionproteins. For purposes of ¹⁸F detection, an antibody or fragmentcontaining a binding site for an antigen associated with a target tissueto be imaged, such as a tumor, may be combined with a second antibody orfragment that binds a hapten on a targetable construct, such as IMP 449,to which a metal-¹⁸F can be attached. The bispecific antibody (DNLconstruct) is administered to a subject, circulating antibody is allowedto clear from the blood and localize to target tissue, and the¹⁸F-labeled targetable construct is added and binds to the localizedantibody for imaging.

Independent transgenic cell lines may be developed for each Fab or IgGfusion protein. Once produced, the modules can be purified if desired ormaintained in the cell culture supernatant fluid. Following production,any DDD₂-fusion protein module can be combined with any correspondingAD-fusion protein module to generate a bispecific DNL construct. Fordifferent types of constructs, different AD or DDD sequences may beutilized. The following DDD sequences are based on the DDD moiety of PKARIIα, while the AD sequences are based on the AD moiety of the optimizedsynthetic AKAP-IS sequence (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445).

DDD1:  (SEQ ID NO: 6) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARADDD2:  (SEQ ID NO: 7) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARAAD1:  (SEQ ID NO: 8) QIEYLAKQIVDNAIQQA AD2:  (SEQ ID NO: 9)CGQIEYLAKQIVDNAIQQAGC

The plasmid vector pdHL2 has been used to produce a number of antibodiesand antibody-based constructs. See Gillies et al., J Immunol Methods(1989), 125:191-202; Losman et al., Cancer (Phila) (1997), 80:2660-6.The di-cistronic mammalian expression vector directs the synthesis ofthe heavy and light chains of IgG. The vector sequences are mostlyidentical for many different IgG-pdHL2 constructs, with the onlydifferences existing in the variable domain (VH and VL) sequences. Usingmolecular biology tools known to those skilled in the art, these IgGexpression vectors can be converted into Fab-DDD or Fab-AD expressionvectors. To generate Fab-DDD expression vectors, the coding sequencesfor the hinge, CH2 and CH3 domains of the heavy chain are replaced witha sequence encoding the first 4 residues of the hinge, a 14 residueGly-Ser linker and the first 44 residues of human RIIα (referred to asDDD1). To generate Fab-AD expression vectors, the sequences for thehinge, CH2 and CH3 domains of IgG are replaced with a sequence encodingthe first 4 residues of the hinge, a 15 residue Gly-Ser linker and a 17residue synthetic AD called AKAP-IS (referred to as AD1), which wasgenerated using bioinformatics and peptide array technology and shown tobind RIIα dimers with a very high affinity (0.4 nM). See Alto, et al.Proc. Natl. Acad. Sci., U.S.A. (2003), 100:4445-50.

Two shuttle vectors were designed to facilitate the conversion ofIgG-pdHL2 vectors to either Fab-DDD1 or Fab-AD1 expression vectors, asdescribed below.

Preparation of CH1

The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as atemplate. The left PCR primer consisted of the upstream (5′) end of theCH1 domain and a SacII restriction endonuclease site, which is 5′ of theCH1 coding sequence. The right primer consisted of the sequence codingfor the first 4 residues of the hinge followed by four glycines and aserine, with the final two codons (GS) comprising a Bam HI restrictionsite. The 410 bp PCR amplimer was cloned into the pGemT PCR cloningvector (Promega, Inc.) and clones were screened for inserts in the T7(5′) orientation.

A duplex oligonucleotide was synthesized by to code for the amino acidsequence of DDD1 preceded by 11 residues of a linker peptide, with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below, with the DDD1 sequence underlined.

(SEQ ID NO: 10) GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRL REARA

Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom, thatoverlap by 30 base pairs on their 3′ ends, were synthesized (SigmaGenosys) and combined to comprise the central 154 base pairs of the 174bp DDD1 sequence. The oligonucleotides were annealed and subjected to aprimer extension reaction with Taq polymerase. Following primerextension, the duplex was amplified by PCR. The amplimer was cloned intopGemT and screened for inserts in the T7 (5′) orientation.

A duplex oligonucleotide was synthesized to code for the amino acidsequence of AD1 preceded by 11 residues of the linker peptide with thefirst two codons comprising a BamHI restriction site. A stop codon andan EagI restriction site are appended to the 3′ end. The encodedpolypeptide sequence is shown below, with the sequence of AD1underlined.

(SEQ ID NO: 11) GSGGGGSGGGGSQIEYLAKQIVDNAIQQA

Two complimentary overlapping oligonucleotides encoding the abovepeptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, weresynthesized and annealed. The duplex was amplified by PCR. The amplimerwas cloned into the pGemT vector and screened for inserts in the T7 (5′)orientation.

Ligating DDD1 with CH1

A 190 bp fragment encoding the DDD1 sequence was excised from pGemT withBamHI and NotI restriction enzymes and then ligated into the same sitesin CH 1-pGemT to generate the shuttle vector CH 1-DDD 1-pGemT.

Ligating AD1 with CH1

A 110 bp fragment containing the AD1 sequence was excised from pGemTwith BamHI and NotI and then ligated into the same sites in CH1-pGemT togenerate the shuttle vector CH1-AD1-pGemT.

Cloning CH1-DDD1 or CH1-AD1 into pdHL2-Based Vectors

With this modular design either CH1-DDD1 or CH1-AD1 can be incorporatedinto any IgG construct in the pdHL2 vector. The entire heavy chainconstant domain is replaced with one of the above constructs by removingthe SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacingit with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excisedfrom the respective pGemT shuttle vector.

Construction of h679-Fd-AD1-pdHL2

h679-Fd-AD1-pdHL2 is an expression vector for production of h679 Fabwith AD1 coupled to the carboxyl terminal end of the CH1 domain of theFd via a flexible Gly/Ser peptide spacer composed of 14 amino acidresidues. A pdHL2-based vector containing the variable domains of h679was converted to h679-Fd-AD1-pdHL2 by replacement of the SacII/EagIfragment with the CH1-AD1 fragment, which was excised from theCH1-AD1-SV3 shuttle vector with SacII and EagI.

Construction of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of astable dimer that comprises two copies of a fusion proteinC-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the carboxylterminus of CH1 via a flexible peptide spacer. The plasmid vectorhMN14(I)-pdHL2, which has been used to produce hMN-14 IgG, was convertedto C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagI restrictionendonucleases to remove the CH1-CH3 domains and insertion of theCH1-DDD1 fragment, which was excised from the CH1-DDD1-SV3 shuttlevector with SacII and EagI.

The same technique has been utilized to produce plasmids for Fabexpression of a wide variety of known antibodies, such as hLL1, hLL2,hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and many others. Generally,the antibody variable region coding sequences were present in a pdHL2expression vector and the expression vector was converted for productionof an AD- or DDD-fusion protein as described above. The AD- andDDD-fusion proteins comprising a Fab fragment of any of such antibodiesmay be combined, in an approximate ratio of two DDD-fusion proteins perone AD-fusion protein, to generate a trimeric DNL construct comprisingtwo Fab fragments of a first antibody and one Fab fragment of a secondantibody.

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production ofC-DDD2-Fab-hMN-14, which possesses a dimerization and docking domainsequence of DDD2 appended to the carboxyl terminus of the Fd of hMN-14via a 14 amino acid residue Gly/Ser peptide linker. The fusion proteinsecreted is composed of two identical copies of hMN-14 Fab held togetherby non-covalent interaction of the DDD2 domains.

Two overlapping, complimentary oligonucleotides, which comprise thecoding sequence for part of the linker peptide and residues 1-13 ofDDD2, were made synthetically. The oligonucleotides were annealed andphosphorylated with T4 PNK, resulting in overhangs on the 5′ and 3′ endsthat are compatible for ligation with DNA digested with the restrictionendonucleases BamHI and PstI, respectively.

The duplex DNA was ligated with the shuttle vector CH1-DDD1-pGemT, whichwas prepared by digestion with BamHI and PstI, to generate the shuttlevector CH1-DDD2-pGemT. A 507 bp fragment was excised from CH1-DDD2-pGemTwith SacII and EagI and ligated with the IgG expression vectorhMN14(I)-pdHL2, which was prepared by digestion with SacII and EagI. Thefinal expression construct was designated C-DDD2-Fd-hMN-14-pdHL2.Similar techniques have been utilized to generated DDD2-fusion proteinsof the Fab fragments of a number of different humanized antibodies.

H679-Fd-AD2-pdHL2

h679-Fab-AD2, was designed to pair as B to C-DDD2-Fab-hMN-14 as A.h679-Fd-AD2-pdHL2 is an expression vector for the production ofh679-Fab-AD2, which possesses an anchor domain sequence of AD2 appendedto the carboxyl terminal end of the CH1 domain via a 14 amino acidresidue Gly/Ser peptide linker. AD2 has one cysteine residue precedingand another one following the anchor domain sequence of AD1.

The expression vector was engineered as follows. Two overlapping,complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprisethe coding sequence for AD2 and part of the linker sequence, were madesynthetically. The oligonucleotides were annealed and phosphorylatedwith T4 PNK, resulting in overhangs on the 5′ and 3′ ends that arecompatible for ligation with DNA digested with the restrictionendonucleases BamHI and SpeI, respectively.

The duplex DNA was ligated into the shuttle vector CH1-AD1-pGemT, whichwas prepared by digestion with BamHI and SpeI, to generate the shuttlevector CH1-AD2-pGemT. A 429 base pair fragment containing CH1 and AD2coding sequences was excised from the shuttle vector with SacII and EagIrestriction enzymes and ligated into h679-pdHL2 vector that prepared bydigestion with those same enzymes. The final expression vector ish679-Fd-AD2-pdHL2.

Example 5 Generation of TF2 DNL Construct

A trimeric DNL construct designated TF2 was obtained by reactingC-DDD2-Fab-hMN-14 with h679-Fab-AD2. A pilot batch of TF2 was generatedwith >90% yield as follows. Protein L-purified C-DDD2-Fab-hMN-14 (200mg) was mixed with h679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio. Thetotal protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA.Subsequent steps involved TCEP reduction, HIC chromatography, DMSOoxidation, and IMP 291 affinity chromatography. Before the addition ofTCEP, SE-HPLC did not show any evidence of a₂b formation. Addition of 5mM TCEP rapidly resulted in the formation of a₂b complex consistent witha 157 kDa protein expected for the binary structure. TF2 was purified tonear homogeneity by IMP 291 affinity chromatography (not shown). IMP 291is a synthetic peptide containing the HSG hapten to which the 679 Fabbinds (Rossi et al., 2005, Clin Cancer Res 11:7122s-29s). SE-HPLCanalysis of the IMP 291 unbound fraction demonstrated the removal of a₄,a₂ and free kappa chains from the product (not shown).

Non-reducing SDS-PAGE analysis demonstrated that the majority of TF2exists as a large, covalent structure with a relative mobility near thatof IgG (not shown). The additional bands suggest that disulfideformation is incomplete under the experimental conditions (not shown).Reducing SDS-PAGE shows that any additional bands apparent in thenon-reducing gel are product-related (not shown), as only bandsrepresenting the constituent polypeptides of TF2 are evident. MALDI-TOFmass spectrometry (not shown) revealed a single peak of 156,434 Da,which is within 99.5% of the calculated mass (157,319 Da) of TF2.

The functionality of TF2 was determined by BIACORE assay. TF2,C-DDD1-hMN-14+h679-AD1 (used as a control sample of noncovalent a₂bcomplex), or C-DDD2-hMN-14+h679-AD2 (used as a control sample ofunreduced a₂ and b components) were diluted to 1 μg/ml (total protein)and passed over a sensorchip immobilized with HSG. The response for TF2was approximately two-fold that of the two control samples, indicatingthat only the h679-Fab-AD component in the control samples would bind toand remain on the sensorchip. Subsequent injections of W12 IgG, ananti-idiotype antibody for hMN-14, demonstrated that only TF2 had aDDD-Fab-hMN-14 component that was tightly associated with h679-Fab-AD asindicated by an additional signal response. The additional increase ofresponse units resulting from the binding of WI2 to TF2 immobilized onthe sensorchip corresponded to two fully functional binding sites, eachcontributed by one subunit of C-DDD2-Fab-hMN-14. This was confirmed bythe ability of TF2 to bind two Fab fragments of WI2 (not shown).

Example 6 Production of TF10 DNL Construct

A similar protocol was used to generate a trimeric TF10 DNL construct,comprising two copies of a C-DDD2-Fab-hPAM4 and one copy ofC-AD2-Fab-679. The TF10 bispecific ([hPAM4]₂×h679) antibody was producedusing the method disclosed for production of the (anti CEA)₂×anti HSGbsAb TF2, as described above. The TF10 construct bears two humanizedPAM4 Fabs and one humanized 679 Fab.

The two fusion proteins (hPAM4-DDD2 and h679-AD2) were expressedindependently in stably transfected myeloma cells. The tissue culturesupernatant fluids were combined, resulting in a two-fold molar excessof hPAM4-DDD2. The reaction mixture was incubated at room temperaturefor 24 hours under mild reducing conditions using 1 mM reducedglutathione. Following reduction, the DNL reaction was completed by mildoxidation using 2 mM oxidized glutathione. TF10 was isolated by affinitychromatography using IMP 291-affigel resin, which binds with highspecificity to the h679 Fab.

Example 7 Sequence Variants for DNL

In certain preferred embodiments, the AD and DDD sequences incorporatedinto the cytokine-MAb DNL complex comprise the amino acid sequences ofAD1 or AD2 and DDD1 or DDD2, as discussed above. However, in alternativeembodiments sequence variants of AD and/or DDD moieties may be utilizedin construction of the DNL complexes. For example, there are only fourvariants of human PKA DDD sequences, corresponding to the DDD moietiesof PKA RIα, RIIα, RIβ and RIIβ. The RIIα DDD sequence is the basis ofDDD1 and DDD2 disclosed above. The four human PKA DDD sequences areshown below. The DDD sequence represents residues 1-44 of RIIα, 1-44 ofRIIβ, 12-61 of RIα and 13-66 of RIα. (Note that the sequence of DDD1 ismodified slightly from the human PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 12)SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEA K PKA RIβ(SEQ ID NO: 13) SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENR QILAPKA RIIα (SEQ ID NO: 14) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQPKA RIIβ (SEQ ID NO: 15) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:6 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 6) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA 

As known in the art, conservative amino acid substitutions have beencharacterized for each of the twenty common L-amino acids. Thus, basedon the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:6are shown in Table 2. In devising Table 2, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. Even withsuch conservative substitutions, there are over twenty million possiblealternative sequences for the 44 residue peptide(2×3×2×2×2×2×2×2×2×2×2×2×2×2×2×4×2×2×2×2×2×4×2×4). The skilled artisanwill realize that an almost unlimited number of alternative specieswithin the genus of DDD moieties can be constructed by standardtechniques, for example using a commercial peptide synthesizer or wellknown site-directed mutagenesis techniques. The effect of the amino acidsubstitutions on AD moiety binding may also be readily determined bystandard binding assays, for example as disclosed in Alto et al. (2003,Proc Natl Acad Sci USA 100:4445-50).

TABLE 2Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 6). Consensussequence disclosed as SEQ ID NO: 16. S H I Q I P P G L T E L L Q G Y T VE V L R T K N A S D N A S D K R Q Q P P D L V E F A V E Y F T R L R E AR A N N E D L D S K K D L K L I I I V V V

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an RII selective AD sequence called AKAP-IS (SEQ ID NO:8), with abinding constant for DDD of 0.4 nM. The AKAP-IS sequence was designed asa peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO:8 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 3 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD1, SEQ ID NO:8), similar tothat shown for DDD1 (SEQ ID NO:6) in Table 2 above.

Even with such conservative substitutions, there are over thirty-fivethousand possible alternative sequences for the 17 residue AD1 (SEQ IDNO:8) peptide sequence (2×3×2×4×3×2×2×2×2×2×2×4). Again, a very largenumber of species within the genus of possible AD moiety sequences couldbe made, tested and used by the skilled artisan, based on the data ofAlto et al. (2003). It is noted that FIG. 2 of Alto (2003) shows an evenlarge number of potential amino acid substitutions that may be made,while retaining binding activity to DDD moieties, based on actualbinding experiments.

AKAP-IS (SEQ ID NO: 8) QIEYLAKQIVDNAIQQA

TABLE 3Conservative Amino Acid Substitutions in AD1 (SEQ ID NO:8). Consensussequence disclosed as SEQ ID NO: 17. Q I E Y L A K Q I V D N A I Q Q A NL D F I R N E Q N N L V T V I S V

Gold (2006, Mol Cell 24:383-95) utilized crystallography and peptidescreening to develop a SuperAKAP-IS sequence (SEQ ID NO:18), exhibitinga five order of magnitude higher selectivity for the RII isoform of PKAcompared with the RI isoform. Underlined residues indicate the positionsof amino acid substitutions, relative to the AKAP-IS sequence, whichincreased binding to the DDD moiety of RIIα. In this sequence, theN-terminal Q residue is numbered as residue number 4 and the C-terminalA residue is residue number 20. Residues where substitutions could bemade to affect the affinity for RIIα were residues 8, 11, 15, 16, 18, 19and 20 (Gold et al., 2006). It is contemplated that in certainalternative embodiments, the SuperAKAP-IS sequence may be substitutedfor the AKAP-IS AD moiety sequence to prepare DNL constructs. Otheralternative sequences that might be substituted for the AKAP-IS ADsequence are shown in SEQ ID NO:19-21. Substitutions relative to theAKAP-IS sequence are underlined. It is anticipated that, as with the AD2sequence shown in SEQ ID NO:9, the AD moiety may also include theadditional N-terminal residues cysteine and glycine and C-terminalresidues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 18) QIEYVAKQIVDYAIHQAAlternative AKAP sequences (SEQ ID NO: 19) QIEYKAKQIVDHAIHQA(SEQ ID NO: 20) QIEYVAKQIVDHAIHQA (SEQ ID NO: 21) QIEYVAKQIVDHAIHQA

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, any of which could be utilized to design a DNLconstruct.

Stokka et al. (2006, Biochem J 400:493-99) also developed peptidecompetitors of AKAP binding to PKA, shown in SEQ ID NO:22-24. Thepeptide antagonists were designated as Ht31 (SEQ ID NO:22), RIAD (SEQ IDNO:23) and PV-38 (SEQ ID NO:24). The Ht-31 peptide exhibited a greateraffinity for the RII isoform of PKA, while the RIAD and PV-38 showedhigher affinity for RI.

Ht31 (SEQ ID NO: 22) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 23)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 24) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006, Biochem J 396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al., reproduced in Table 4 below. AKAPIS represents asynthetic RII subunit-binding peptide. All other peptides are derivedfrom the RII-binding domains of the indicated AKAPs.

TABLE 4 AKAP Peptide sequences Peptide Sequence AKAPIS QIEYLAKQIVDNAIQQA(SEQ ID NO: 8) AKAPIS-P QIEYLAKQIPDNAIQQA (SEQ ID NO: 25) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 26) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 27) AKAP7δ-wt-PEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 28) pep AKAP7δ-PEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 29) L304T-pep AKAP7δ-PEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 30) L308D-pep AKAP7δ-P-PEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 31) pep AKAP7δ-PP-PEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 32) pep AKAP7δ-PEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 33) L314E-pep AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 34) AKAP2-pepLVDDPLEYQAGLLVQNA1QQAIAEQ (SEQ ID NO: 35) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 36) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 37) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 38) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 39) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 40) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 41) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 42)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:8). The residues are the same as observed byAlto et al. (2003), with the addition of the C-terminal alanine residue.(See FIG. 4 of Hundsrucker et al. (2006), incorporated herein byreference.) The sequences of peptide antagonists with particularly highaffinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS (SEQ ID NO: 8) QIEYLAKQIVDNAIQQA

Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA. RIIα DDD sequence of SEQ ID NO:6. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized.

(SEQ ID NO: 6) SHIQ IP P GL TELLQGYT V EVLR Q QP P DLVEFA VE YF TR L REAR A

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:6) sequence, based on the data of Carr et al. (2001) is shownin Table 5. Even with this reduced set of substituted sequences, thereare over 65,000 possible alternative DDD moiety sequences that may beproduced, tested and used by the skilled artisan without undueexperimentation. The skilled artisan could readily derive suchalternative DDD amino acid sequences as disclosed above for Table 2 andTable 3.

TABLE 5Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO:6). Consensussequence disclosed as SEQ ID NO: 43. S H I Q I P P G L T E L L Q G Y T VE V L R T N S I L A Q Q P P D L V E F A V E Y F T R L R E A R A N I D SK K L L L I I A V V

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Example 8 In Vivo Imaging Using ¹⁸F-Labeled Peptides and Comparison with¹⁸F-FDG

In vivo imaging techniques using pretargeting with bispecific antibodiesand labeled targeting peptides were used to successfully detect tumorsof relatively small size. The ¹⁸F was purified on a WATERS® ACCELL™ PlusQMA Light cartridge. The ¹⁸F⁻ eluted with 0.4 M KHCO₃ was mixed with 3μL 2 mM Al³⁺ in a pH 4 acetate buffer. The Al¹⁸F solution was theninjected into the ascorbic acid IMP449 labeling vial and heated to 105°C. for 15 min. The reaction solution was cooled and mixed with 0.8 mL DIwater. The reaction contents were loaded on a WATERS® OASIS® 1 cc HLBColumn and eluted with 2×200 μL 1:1 EtOH/H₂O. TF2 was prepared asdescribed above. TF2 binds divalently to carcinoembryonic antigen (CEA)and monovalently to the synthetic hapten, HSG(histamine-succinyl-glycine).

Biodistribution and microPET Imaging.

Six-week-old NCr nu-m female nude mice were implanted s.c. with thehuman colonic cancer cell line, LS 174T (ATCC, Manassas, Va.). Whentumors were visibly established, pretargeted animals were injectedintravenously with 162 μg (˜1 nmole/0.1 mL) TF2 or TF10 (controlnon-targeting tri-Fab bsMAb), and then 16-18 h later, ˜0.1 nmol ofAl¹⁸F(IMP449) (84 μCi, 3.11 MBq/0.1 mL) was injected intravenously.Other non-pretargeted control animals received ¹⁸F alone (150 μCi, 5.5MBq), Al¹⁸F complex alone (150 μCi, 5.55 MBq), the Al¹⁸F(IMP449) peptidealone (84 μCi, 3.11 MBq), or ¹⁸F-FDG (150 μCi, 5.55 MBq). ¹⁸F and¹⁸F-FDG were obtained on the day of use from IBA Molecular (Somerset,N.J.). Animals receiving ¹⁸F-FDG were fasted overnight, but water wasgiven ad libitum.

At 1.5 h after the radiotracer injection, animals were anesthetized,bled intracardially, and necropsied. Tissues were weighed and countedtogether with a standard dilution prepared from each of the respectiveproducts. Due to the short physical half-life of ¹⁸F, standards wereinterjected between each group of tissues from each animal. Uptake inthe tissues is expressed as the counts per gram divided by the totalinjected activity to derive the percent-injected dose per gram (% ID/g).

Two types of imaging studies were performed. In one set, 3 nude micebearing small LS174T subcutaneous tumors received either the pretargetedAl¹⁸F(IMP449), Al¹⁸F(IMP449) alone (not pretargeted), both at 135 μCi (5MBq; 0.1 nmol), or ¹⁸F-FDG (135 μCi, 5 MBq). At 2 h after theintravenous radiotracer injection, the animals were anesthetized with amixture of O₂/N₂O and isoflurane (2%) and kept warm during the scan,performed on an INVEON® animal PET scanner (Siemens PreclinicalSolutions, Knoxville, Tenn.).

Representative coronal cross-sections (0.8 mm thick) in a plane locatedapproximately in the center of the tumor were displayed, withintensities adjusted until pixel saturation occurred in any region ofthe body (excluding the bladder) and without background adjustment.

In a separate dynamic imaging study, a single LS174T bearing nude mousethat was given the TF2 bsMAb 16 h earlier was anesthetized with amixture of O₂/N₂O and isoflurane (2%), placed supine on the camera bed,and then injected intravenously with 219 μCi (8.1 MBq) Al¹⁸F(IMP449)(0.16 nmol). Data acquisition was immediately initiated over a period of120 minutes. The scans were reconstructed using OSEM3D/MAP. Forpresentation, time-frames ending at 5, 15, 30, 60, 90, and 120 min weredisplayed for each cross-section (coronal, sagittal, and transverse).For sections containing tumor, at each interval the image intensity wasadjusted until pixel saturation first occurred in the tumor. Imageintensity was increased as required over time to maintain pixelsaturation within the tumor. Coronal and sagittal cross-sections withouttumor taken at the same interval were adjusted to the same intensity asthe transverse section containing the tumor. Background activity was notadjusted.

Results

While ¹⁸F alone and [Al¹⁸F] complexes had similar uptake in all tissues,considerable differences were found when the complex was chelated toIMP449 (Table 6). The most striking differences were found in the uptakein the bone, where the non-chelated ¹⁸F was 60- to nearly 100-foldhigher in the scapula and ˜200-fold higher in the spine. Thisdistribution is expected since ¹⁸F, or even a metal-fluoride complex, isknown to accrete in bone (Franke et al. 1972, Radiobiol. Radiother.(Berlin) 13:533). Higher uptake was also observed in the tumor andintestines as well as in muscle and blood. The chelated Al¹⁸F(IMP449)had significantly lower uptake in all the tissues except the kidneys,illustrating the ability of the chelate-complex to be removedefficiently from the body by urinary excretion.

Pretargeting the Al¹⁸F(IMP449) using the TF2 anti-CEA bsMAb shifteduptake to the tumor, increasing it from 0.20±0.05 to 6.01±1.72% injecteddose per gram at 1.5 h, while uptake in the normal tissues was similarto the Al¹⁸F(IMP449) alone. Tumor/nontumor ratios were 146±63, 59±24,38±15, and 2.0±1.0 for the blood, liver, lung, and kidneys,respectively, with other tumor/tissue ratios >100:1 at this time.Although both ¹⁸F alone and [Al¹⁸F] alone had higher uptake in the tumorthan the chelated Al¹⁸F(IMP449), yielding tumor/blood ratios of 6.7±2.7and 11.0±4.6 vs. 5.1±1.5, respectively, tumor uptake and tumor/bloodratios were significantly increased with pretargeting (all P values<0.001).

Biodistribution was also compared to the most commonly used tumorimaging agent, [¹⁸F]FDG, which targets tissues with high glucoseconsumption and metabolic activity (Table 6). Its uptake was appreciablyhigher than the Al¹⁸F(IMP449) in all normal tissues, except the kidney.Tumor uptake was similar for both the pretargeted Al¹⁸F(IMP449) and¹⁸F-FDG, but because of the higher accretion of [¹⁸F]FDG in most normaltissues, tumor/nontumor ratios with ¹⁸F-FDG were significantly lowerthan those in the pretargeted animals (all P values <0.001).

TABLE 6 Biodistribution of TF2-pretargeted Al¹⁸F(IMP449) and othercontrol ¹⁸F-labeled agents in nude mice bearing LS174T human colonicxenografts. For pretargeting, animals were given TF2 16 h before theinjection of the Al¹⁸F(IMP449). All injections were administeredintravenously. Percent Injected Dose Per Gram (Mean ± SD) at 1.5 hrPost-Injection Al¹⁸F(IMP449) TF2-pretargeted ¹⁸F alone [Al¹⁸F] alonealone Al¹⁸F(IMP449) ¹⁸F-FDG Tumor 1.02 ± 0.45 1.38 ± 0.39 0.20 ± 0.056.01 ± 1.72 7.25 ± 2.54 Liver 0.11 ± 0.02 0.12 ± 0.02 0.08 ± 0.03 0.11 ±0.03 1.34 ± 0.36 Spleen 0.13 ± 0.06 0.10 ± 0.03 0.08 ± 0.02 0.08 ± 0.022.62 ± 0.73 Kidney 0.29 ± 0.07 0.25 ± 0.07 3.51 ± 0.56 3.44 ± 0.99 1.50± 0.61 Lung 0.26 ± 0.08 0.38 ± 0.19 0.11 ± 0.03 0.17 ± 0.04 3.72 ± 1.48Blood 0.15 ± 0.03 0.13 ± 0.03 0.04 ± 0.01 0.04 ± 0.02 0.66 ± 0.19Stomach 0.21 ± 0.13 0.15 ± 0.05 0.20 ± 0.32 0.12 ± 0.18 2.11 ± 1.04Small Int. 1.53 ± 0.33 1.39 ± 0.34 0.36 ± 0.23 0.27 ± 0.10 1.77 ± 0.61Large Int. 1.21 ± 0.13 1.78 ± 0.70 0.05 ± 0.04 0.03 ± 0.01 2.90 ± 0.79Scapula 6.13 ± 1.33 9.83 ± 2.31 0.08 ± 0.06 0.04 ± 0.02 10.63 ± 5.88 Spine 19.88 ± 2.12  19.03 ± 2.70  0.13 ± 0.14 0.08 ± 0.03 4.21 ± 1.79Muscle 0.16 ± 0.05 0.58 ± 0.36 0.06 ± 0.05 0.10 ± 0.20 4.35 ± 3.01 Brain0.15 ± 0.06 0.13 ± 0.03 0.01 ± 0.01 0.01 ± 0.00 10.71 ± 4.53  Tumor wt(g) 0.29 ± 0.07 0.27 ± 0.10 0.27 ± 0.08 0.33 ± 0.11 0.25 ± 0.21 N 6 7 87 5

Several animals were imaged to further analyze the biodistribution ofAl¹⁸F(IMP449) alone or Al¹⁸F(MP449) pretargeted with TF2, as well[¹⁸F]FDG. Static images initiated at 2.0 h after the radioactivity wasinjected corroborated the previous tissue distribution data showinguptake almost exclusively in the kidneys (FIG. 1). A 21-mg tumor waseasily visualized in the pretargeted animal, while the animal given theAl¹⁸F(IMP449) alone failed to localize the tumor, having only renaluptake. No evidence of bone accretion was observed, suggesting that theAl¹⁸F was bound firmly to IMP 449. This was confirmed in anotherpretargeted animal that underwent a dynamic imaging study that monitoredthe distribution of the Al¹⁸F(IMP449) in 5-min intervals over 120minutes (FIG. 2). Coronal and sagittal slices showed primarily cardiac,renal, and some hepatic uptake over the first 5 min, but heart and liveractivity decreased substantially over the next 10 min, while the kidneysremained prominent throughout the study. There was no evidence ofactivity in the intestines or bone over the full 120-min scan. Uptake ina 35-mg LS174T tumor was first observed at 15 min, and by 30 min, thesignal was very clearly delineated from background, with intense tumoractivity being prominent during the entire 120-min scanning.

In comparison, static images from an animal given ¹⁸F-FDG showed theexpected pattern of radioactivity in the bone, heart muscle, and brainobserved previously (McBride et al., 2006, J. Nucl. Med. 47:1678;Sharkey et al., 2008, Radiology 246:497), with considerably morebackground activity in the body (FIG. 1). Tissue uptake measured in the3 animals necropsied at the conclusion of the static imaging studyconfirmed much higher tissue ¹⁸F radioactivity in all tissues (notshown). While tumor uptake with ¹⁸F-FDG was higher in this animal thanin the pretargeted one, tumor/blood ratios were more favorable forpretargeting; and with much less residual activity in the body, tumorvisualization was enhanced by pretargeting.

These studies demonstrate that a hapten-peptide used in pretargetedimaging can be rapidly labeled (60 min total preparation time) with ¹⁸Fby simply forming an aluminum-fluoride complex that can then be bound bya suitable chelate and incorporated into the hapten-peptide. This can bemade more general by simply coupling the [Al¹⁸F]-chelate to any moleculethat can be attached to the chelating moiety and be subsequentlypurified.

This report describes a direct, facile, and rapid method of binding ¹⁸Fto various compounds via an aluminum conjugate. The [Al¹⁸F] peptide wasstable in vitro and in vivo when bound by a NOTA-based chelate. Yieldswere within the range found with conventional ¹⁸F labeling procedures.These results further demonstrate the feasibility of PET imaging usingmetal¹⁸F chelated to a wide variety of targeting molecules.

Example 9 Preparation and Labeling of IMP460 with Al—¹⁸F

IMP460 NODA-Ga-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (SEQ ID NO:44) waschemically synthesized. The NODA-Ga ligand was purchased from CHEMATECH®and attached on the peptide synthesizer like the other amino acids. Thepeptide was synthesized on Sieber amide resin with the amino acids andother agents added in the following order Aloc-D-Lys(Fmoc)-OH,Trt-HSG-OH, Aloc removal, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH,Trt-HSG-OH, Aloc removal, Fmoc-D-Ala-OH, and NODA-GA(tBu)₃. The peptidewas then cleaved and purified by HPLC to afford the product.

Radiolabeling of IMP460

IMP 460 (0.0020 g) was dissolved in 732 μL, pH 4, 0.1 M NaOAc. The ¹⁸Fwas purified as described above, neutralized with glacial acetic acidand mixed with the Al solution. The peptide solution, 20 μL was thenadded and the solution was heated at 99° C. for 25 min. The crudeproduct was then purified on a WATERS® HLB column. The [Al¹⁸F] labeledpeptide was in the 1:1 EtOH/H₂O column eluent. The reverse phase HPLCtrace in 0.1% TFA buffers showed a clean single HPLC peak at theexpected location for the labeled peptide (not shown).

Example 10 Synthesis and Labeling of IMP461 and IMP462 NOTA-ConjugatedPeptides

The simplest possible NOTA ligand (protected for peptide synthesis) wasprepared and incorporated into two peptides for pretargeting—IMP461 andIMP462.

Synthesis of Bis-t-butyl-NOTA

NO2AtBu (0.501 g 1.4×10⁻³ mol) was dissolved in 5 mL anhydrousacetonitrile. Benzyl-2-bromoacetate (0.222 mL, 1.4×10⁻³ mol) was addedto the solution followed by 0.387 g of anhydrous K₂CO₃. The reaction wasallowed to stir at room temperature overnight. The reaction mixture wasfiltered and concentrated to obtain 0.605 g (86% yield) of the benzylester conjugate. The crude product was then dissolved in 50 mL ofisopropanol, mixed with 0.2 g of 10% Pd/C (under Ar) and placed under 50psi H₂ for 3 days. The product was then filtered and concentrated undervacuum to obtain 0.462 g of the desired product ESMS [M−H]⁻ 415.

Synthesis of IMP461

The peptide was synthesized on Sieber amide resin with the amino acidsand other agents added in the following order Aloc-D-Lys(Fmoc)-OH,Trt-HSG-OH, Aloc removal, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH,Trt-HSG-OH, Aloc removal, Fmoc-D-Ala-OH, and Bis-t-butyl NOTA. Thepeptide was then cleaved and purified by HPLC to afford the productIMP461 ESMS MH⁺ 1294 NOTA-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂; SEQ IDNO:45).

Synthesis of IMP 462

The peptide was synthesized on Sieber amide resin with the amino acidsand other agents added in the following order Aloc-D-Lys(Fmoc)-OH,Trt-HSG-OH, Aloc removal, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH,Trt-HSG-OH, Aloc removal, Fmoc-D-Asp(But)-OH, and Bis-t-butyl NOTA. Thepeptide was then cleaved and purified by HPLC to afford the productIMP462 ESMS MH⁺ 1338 (NOTA-D-Asp-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂; SEQ IDNO:46).

¹⁸F Labeling of IMP461 & IMP462

The peptides were dissolved in pH 4.13, 0.5 M NaOAc to make a 0.05 Mpeptide solution, which was stored in the freezer until needed. The F-18was received in 2 mL of water and trapped on a SEP-PAK® Light, WATERS®ACCELL™ Plus QMA Cartridge. The ¹⁸F was eluted from the column with 200μL aliquots of 0.4 M KHCO₃. The bicarbonate was neutralized to ˜pH 4 bythe addition of 10 μL of glacial acetic acid to the vials before theaddition of the activity. A 100 μL aliquot of the purified ¹⁸F solutionwas removed and mixed with 3 μL, 2 mM Al in pH 4, 0.1 M NaOAc. Thepeptide, 10 μL (0.05 M) was added and the solution was heated at ˜100°C. for 15 mM. The crude reaction mixture was diluted with 700 μL DIwater and placed on an HLB column and after washing the ¹⁸F was elutedwith 2×100 μL of 1:1 EtOH/H₂O to obtain the purified ¹⁸F-labeledpeptide.

Example 11 Preparation and ¹⁸F-Labeling of IMP467

(SEQ ID NO: 47) IMP- C-NETA-succinyl-D-Lys(HSG)-D-Tyr-D-Lys(HSG)- 467NH₂

Tetra tert-butyl C-NETA-succinyl was produced. The tert-Butyl{4-[2-(Bis-(tert-butyoxycarbonyl)methyl-3-(4-nitrophenyl)propyl]-7-tert-butyoxycarbonyl[1,4,7]triazanonan-1-yl}was prepared as described in Chong et al. (J. Med. Chem. 2008,51:118-125).

The peptide, IMP467 C-NETA-succinyl-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH₂ (SEQID NO:47) was made on Sieber Amide resin by adding the following aminoacids to the resin in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH,the Aloc was cleaved Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH,Trt-HSG-OH, the Aloc was cleaved, tert-Butyl{4-[Bis-(tert-butoxycarbonylmethyl)amino)-3-(4-succinylamidophenyl)propyl]-7-tert-butoxycarbonylmethyl[1,4,7]triazanonan-1-yl}acetate.The peptide was then cleaved from the resin and purified by RP-HPLC toyield 6.3 mg of IMP467. The crude peptide was purified by highperformance liquid chromatography (HPLC) using a C18 column.

Radiolabeling

A 2 mM solution of IMP467 was prepared in pH 4, 0.1 M NaOAc. The ¹⁸F⁻,139 mCi, was eluted through a WATERS® ACCELL™ Plus SEP-PAK® Light QMAcartridge and the ¹⁸F⁻ was eluted with 1 mL of 0.4 M KHCO₃. The labeledIMP467 was purified by HLB RP-HPLC. The RP-HPLC showed two peaks eluting(not shown), which are believed to be diastereomers of Al¹⁸F(IMP467).Supporting this hypothesis, there appeared to be some interconversionbetween the two HLB peaks when IMP467 was incubated at 37° C. (notshown). In pretargeting techniques as discussed below, since the[Al¹⁸F]-chelator complex is not part of the hapten site for antibodybinding, the presence of diastereomers does not appear to affecttargeting of the ¹⁸F-labeled peptide to diseased tissues.

Comparison of Yield of Radiolabeled Peptides

In an attempt to improve labeling yields while maintaining in vivostability, 3 NOTA derivatives of pretargeting peptide were synthesized(IMP460, IMP461 and IMP467). Of these, IMP467 nearly doubled thelabeling yields of the other peptides (Table 7). All of the labelingstudies in Table 7 were performed with the same number of moles ofpeptide and aluminum. The results shown in Table 7 represent anexemplary labeling experiment with each peptide.

The ¹⁸F-labeling yield of IMP467 was ˜70% when only 40 nmol (˜13-foldless than IMP449) was used with 1.3 GBq (35 mCi) of ¹⁸F, indicating thisligand has improved binding properties for the Al¹⁸F complex. Byenhancing the kinetics of ligand binding, yields were substantiallyimproved (average 65-75% yield), while using fewer moles of IMP467 (40nmol), relative to IMP449 (520 nmol, 44% yield).

TABLE 7 Comparison of yields of different NOTA containing peptidesPeptide Yield IMP449 44% IMP460 5.8%  IMP461 31% IMP467 87%

Example 12 Factors Affecting Yield and Stability of IMP467 Labeling

Peptide Concentration

To examine the effect of varying peptide concentration on yield, theamount of binding of Al¹⁸F to peptide was determined in a constantvolume (63 μL) with a constant amount of Al³⁺ (6 nmol) and ¹⁸F, butvarying the amount of peptide added. The yield of labeled peptide IMP467decreased with a decreasing concentration of peptide as follows: 40 nmolpeptide (82% yield); 30 nmol (79% yield); 20 nmol (75% yield); 10 nmol(49% yield). Thus, varying the amount of peptide between 20 and 40 nmolhad little effect on yield with IMP467. However, a decreased yield wasobserved starting at 10 nmol of peptide in the labeling mix.

Aluminum Concentration

When IMP467 was labeled in the presence of increasing amounts of Al³⁺(0, 5, 10, 15, 20 of 2 mM Al in pH 4 acetate buffer and keeping thetotal volume constant), yields of 3.5%, 80%, 77%, 78% and 74%,respectively, were achieved. These results indicated that (a)non-specific binding of ¹⁸F to this peptide in the absence of Al³⁺ isminimal, (b) 10 nmol of Al³⁺ was sufficient to allow for maximum¹⁸F-binding, and (c) higher amounts of Al³⁺ did not reduce bindingsubstantially, indicating that there was sufficient chelation capacityat this peptide concentration.

Kinetics of Al¹⁸F(IMP467) Radiolabeling

Kinetic studies showed that binding was complete within 5 min at 107° C.(5 min, 68%; 10 min, 61%; 15 min, 71%; and 30 min, 75%) with onlymoderate increases in isolated yield with reaction times as long as 30min. A radiolabeling reaction of IMP467 performed at 50° C. showed thatno binding was achieved at the lower temperature. Additionalexperiments, disclosed in the Examples below, show that under someconditions a limited amount of labeling can occur at reducedtemperatures.

Effect of pH

The optimal pH for labeling was between 4.3 and 5.5. Yield ranged from54% at pH 2.88; 70-77% at pH 3.99; 70% at pH 5; 41% at pH 6 to 3% at pH7.3. The process could be expedited by eluting the ¹⁸F⁻ from the anionexchange column with nitrate or chloride ion instead of carbonate ion,which eliminates the need for adjusting the eluent to pH 4 with glacialacetic acid before mixing with the AlCl₃.

High-Dose Radiolabeling of IMP467

Five microliters of 2 mM Al³⁺ stock solution were mixed with 50 μL of¹⁸F 1.3 GBq (35 mCi) followed by the addition of 20 μL of 2 mM IMP467 in0.1 mM, pH 4.1 acetate buffer. The reaction solution was heated to 104°C. for 15 min and then purified on an HLB column (˜10 min) as describedabove, isolating 0.68 GBq (18.4 mCi) of the purified peptide in 69%radiochemical yield with a specific activity of 17 GBq/μmol (460Ci/mmol). The reaction time was 15 min and the purification time was 12min. The reaction was started 10 min after the 1.3 GBq (35 mCi)¹⁸F⁻ waspurified, so the total time from the isolation of the ¹⁸F⁻ to thepurified final product was 37 min with a 52% yield without correctingfor decay.

Human Serum Stability Test

An aliquot of the HLB purified peptide (˜30 μL) was diluted with 200 μLhuman serum (previously frozen) and placed in the 37° C. HPLC samplechamber. Aliquots were removed at various time points and analyzed byHPLC. The HPLC analysis showed very high stability of the ¹⁸F-labeledpeptides in serum at 37° C. for at least five hours (not shown). Therewas no detectable breakdown of the ¹⁸F-labeled peptide after a five hourincubation in serum (not shown).

The IMP461 and 11\0462 ligands have two carboxyl groups available tobind the aluminum whereas the NOTA ligand in IMP467 had four carboxylgroups. The serum stability study showed that the complexes with IMP467were stable in serum under conditions replicating in vivo use. In vivobiodistribution studies with labeled IMP467 show that the Al¹⁸F-labeledpeptide is stable under actual in vivo conditions (not shown).

Peptides can be labeled with ¹⁸F rapidly (30 min) and in high yield byforming Al¹⁸F complexes that can be bound to a NOTA ligand on a peptideand at a specific activity of at least 17 GBq/μmol, without requiringHPLC purification. The Al¹⁸F(NOTA)-peptides are stable in serum and invivo. Modifications of the NOTA ligand can lead to improvements in yieldand specific activity, while still maintaining the desired in vivostability of the Al¹⁸F(NOTA) complex, and being attached to ahydrophilic linker aids in the renal clearance of the peptide. Further,this method avoids the dry-down step commonly used to label peptideswith ¹⁸F. As shown in the following Examples, this new ¹⁸F-labelingmethod is applicable to labeling of a broad spectrum of targetingpeptides.

Optimized Labeling of Al¹⁸F(IMP467)

Optimized conditions for ¹⁸F-labeling of IMP467 were identified. Theseconsisted of eluting ¹⁸F⁻ with commercial sterile saline (pH 5-7),mixing with 20 nmol of AlCl₃ and 40 nmol IMP467 in pH 4 acetate bufferin a total volume of 100 μL, heating to 102° C. for 15 min, andperforming SPE separation. High-yield (85%) and high specific activity(115 GBq/μmol) were obtained with IMP467 in a single step, 30-minprocedure after a simple solid-phase extraction (SPE) separation withoutthe need for HPLC purification. Al¹⁸F(IMP467) was stable in PBS or humanserum, with 2% loss of ¹⁸F⁻ after incubation in either medium for 6 h at37° C.

Concentration and Purification of ¹⁸F⁻

Radiochemical-grade ¹⁸F⁻ needs to be purified and concentrated beforeuse. We examined 4 different SPE purification procedures to process the¹⁸F⁻ prior to its use. Most of the radiolabeling procedures wereperformed using ¹⁸F⁻ prepared by a conventional process. The ¹⁸F⁻ in 2mL of water was loaded onto a SEP-PAK® Light, Waters Accell™ QMA PlusCartridge that was pre-washed with 10 mL of 0.4M KHCO₃, followed by 10mL water. After loading the ¹⁸F⁻ onto the cartridge, it was washed with5 mL water to remove any dissolved metal and radiometal impurities. Theisotope was then eluted with ˜1 mL of 0.4M KHCO₃ in several fractions toisolate the fraction with the highest concentration of activity. Theeluted fractions were neutralized with 5 μL of glacial acetic acid per100 μL, of solution to adjust the eluent to pH 4-5.

In the second process, the QMA cartridge was washed with 10 mL pH 8.4,0.5 M NaOAc followed by 10 mL DI H₂O. ¹⁸F⁻ was loaded onto the column asdescribed above and eluted with 1 mL, pH 6, 0.05 M KNO₃ in 200-μLfractions with 60-70% of the activity in one of the fractions. No pHadjustment of this solution was needed.

In the third process, the QMA cartridge was washed with 10 mL pH 8.4,0.5 M NaOAc followed by 10 mL DI H₂O. The ¹⁸F⁻ was loaded onto thecolumn as described above and eluted with 1 mL, pH 5-7, 0.154 Mcommercial normal saline in 200-μL fractions with 80% of the activity inone of the fractions. No pH adjustment of this solution was needed.

Finally, we devised a method to prepare a more concentrated andhigh-activity ¹⁸F⁻ solution, using tandem ion exchange. Briefly, Tygontubing (1.27 cm long, 0.64 cm OD) was inserted into a TRICORN™ 5/20column and filled with ˜200 μL of AG 1-X8 resin, 100-200 mesh. The resinwas washed with 6 mL 0.4 M K₂CO₃ followed by 6 mL H₂O. A SEP-PAK® lightWaters ACCELL™ Plus CM cartridge was washed with DI H₂O. Using a syringepump, the crude ¹⁸F⁻ that was received in 5-mL syringe in 2 mL DI H₂Oflowed slowly through the CM cartridge and the TRICORN™ column over ˜5min followed by a 6 mL wash with DI H₂O through both ion-bindingcolumns. Finally, 0.4 M K₂CO₃ was pushed through only the TRICORN™column in 50-μL fractions. Typically, 40 to 60% of the eluted activitywas in one 50-μL fraction. The fractions were collected in 2.0 mLfree-standing screw-cap microcentrifuge tubes containing 5 μL glacialacetic acid to neutralize the carbonate solution. The elution vial withthe most activity was then used as the reaction vial.

Example 13 Labeling by Addition of ¹⁸F⁻ to a Peptide Complexed withAluminum

An HSG containing peptide (IMP 465,Al(NOTA)-D-Ala-D-Lys(HSG)-D-T-D-Lys(HSG)-NH₂) (SEQ ID NO:48) linked tomacrocyclic NOTA complexed with aluminum, was successfully labeled withF-18. ¹⁸F incorporation using 40 nmol of IMP 465 was 13.20%. Anintermediate peptide, IMP 461, was made as described above. Then 25.7 mgof IMP461 was dissolved in 2 mL DI water to which was added 10.2 mgAlCl₃.3H₂O and the resultant solution heated to 100° C. for 1 h. Thecrude reaction mixture was purified by RP-HPLC to yield 19.6 mg ofIMP465.

For ¹⁸F-labeling, 50 μL ¹⁸F solution [0.702 mCi of ¹⁸F⁻] and 20 μL (40nmol) 2 mM IMP465 solution (0.1 M NaOAc, pH 4.18) was heated to 101° C.for 17 minutes. Reverse Phase HPLC analysis showed 15.38% (RT about 8.60min) of the activity was attached to the peptide and 84.62% of theactivity eluted at the void volume of the column (2.60 min).

In a separate experiment, the percent yield of ¹⁸F-labeled peptide couldbe improved by varying the amount of peptide added. The percent yieldobserved for IMP465 was 0.27% at 10 nmol peptide, 1.8% at 20 nmol ofpeptide and 49% at 40 nmol of peptide.

IMP467 showed higher yield than IMP461 when peptide was pre-incubatedwith aluminum before exposure to ¹⁸F. IMP467 was incubated with aluminumat room temperature and then frozen and lyophilized. The amount ofaluminum added for the pre-incubation was varied.

TABLE 8 Labeling of IMP467 Pre-Incubated with Aluminum Before ¹⁸F⁻ isAdded Isolated IMP467 + Al Premixed, Frozen and Lyophilized LabelingYield 40 nmol IMP467 + 10 nmol Al Premix 82% 40 nmol IMP467 + 20 nmol AlPremix 64% 40 nmol IMP467 + 30 nmol Al Premix 74% 40 nmol IMP467 + 6nmol Al Normal Labeling 77% (Mix Al + ¹⁸F first)

The yields were comparable to those obtained when IMP467 is labeled byaddition of an Al¹⁸F complex. Thus, ¹⁸F labeling by addition of ¹⁸F to apeptide with aluminum already bound to the chelating moiety is afeasible alternative approach to pre-incubating the metal with ¹⁸F⁻prior to addition to the chelating moiety.

Example 14 Synthesis and Labeling of IMP468 Bombesin Peptide

The ¹⁸F labeled targeting moieties are not limited to antibodies orantibody fragments, but rather can include any molecule that bindsspecifically or selectively to a cellular target that is associated withor diagnostic of a disease state or other condition that may be imagedby ¹⁸F PET. Bombesin is a 14 amino acid peptide that is homologous toneuromedin B and gastrin releasing peptide, as well as a tumor markerfor cancers such as lung and gastric cancer and neuroblastoma. IMP468(NOTA-NH—(CH₂)₇CO-Gln-Trp-Val-Trp-Ala-Val-Gly-His-Leu-Met-NH₂; SEQ IDNO:49) was synthesized as a bombesin analogue and labeled with ¹⁸F totarget the gastrin-releasing peptide receptor.

The peptide was synthesized by Fmoc based solid phase peptide synthesison Sieber amide resin, using a variation of a synthetic scheme reportedin the literature (Prasanphanich et al., 2007, PNAS USA 104:12463-467).The synthesis was different in that a bis-t-butyl NOTA ligand was add tothe peptide during peptide synthesis on the resin.

IMP468 (0.0139 g, 1.02×10⁻⁵ mol) was dissolved in 203 μL of 0.5 M pH4.13 NaOAc buffer. The peptide dissolved but formed a gel on standing sothe peptide gel was diluted with 609 μL of 0.5 M pH 4.13 NaOAc bufferand 406 μL of ethanol to produce an 8.35×10⁻³ M solution of the peptide.The ¹⁸F was purified on a QMA cartridge and eluted with 0.4 M KHCO₃ in200 μL fractions, neutralized with 10 μL of glacial acetic acid. Thepurified ¹⁸F, 40 μL, 1.13 mCi was mixed with 3 μL of 2 mM AlCl₃ in pH 4,0.1 M NaOAc buffer. IMP468 (59.2 μL, 4.94×10⁻⁷ mol) was added to theAl¹⁸F solution and placed in a 108° C. heating block for 15 min. Thecrude product was purified on an HLB column, eluted with 2×200 μL of 1:1EtOH/H₂O to obtain the purified ¹⁸F-labeled peptide in 34% yield.

Example 15 Imaging of Tumors Using ¹⁸F Labeled Bombesin

A NOTA-conjugated bombesin derivative (IMP468) was prepared as describedabove. We began testing its ability to block radiolabeled bombesin frombinding to PC-3 cells as was done by Prasanphanich et al. (PNAS104:12462-12467, 2007). Our initial experiment was to determine ifIMP468 could specifically block bombesin from binding to PC-3 cells. Weused IMP333 as a non-specific control. In this experiment, 3×10⁶ PC-3cells were exposed to a constant amount (˜50,000 cpms) of ¹²⁵I-Bombesin(Perkin-Elmer) to which increasing amounts of either IMP468 or IMP333was added. A range of 56 to 0.44 nM was used as our inhibitoryconcentrations.

The results showed that we could block the binding of ¹²⁵I-BBN withIMP468 but not with the control peptide (IMP333) (not shown), thusdemonstrating the specificity of IMP468. Prasanphanich indicated an IC₅₀for their peptide at 3.2 nM, which is approximately 7-fold lower thanwhat we found with IMP468 (21.5 nM).

This experiment was repeated using a commercially available BBN peptide.We increased the amount of inhibitory peptide from 250 to 2 nM to blockthe ¹²⁵I-BBN from binding to PC-3 cells. We observed very similarIC₅₀-values for IMP468 and the BBN positive control with an IC₅₀-valuehigher (35.9 nM) than what was reported previously (3.2 nM) but close towhat the BBN control achieved (24.4 nM).

To examine in vivo targeting, the distribution of Al¹⁸F(IMP468) wasexamined in scPC3 prostate cancer xenograft bearing nude male mice;alone vs. blocked with bombesin. For radiolabeling, aluminum chloride(10 μL, 2 mM), 51.9 mCi of ¹⁸F (from QMA cartridge), acetic acid, and 60μL, of IMP468 (8.45 mM in ethanol/NaOAc) were heated at 100° C. for 15min. The reaction mixture was purified on reverse phase HPLC. Fractions40 and 41 (3.56, 1.91 mCi) were pooled and applied to HLB column forsolvent exchange. The product was eluted in 800 μL (3.98 mCi) and 910μCi remained on the column. iTLC developed in saturated NaCl showed 0.1%unbound activity.

A group of six tumor-bearing mice were injected with Al¹⁸F(IMP468) (167μCi, ˜9×10⁻¹⁰ mol) and necropsied 1.5 h later. Another group of six micewere injected iv with 100 μg (6.2×10⁻⁸ mol) of bombesin 18 min beforeadministering Al¹⁸F(IMP468). The second group was also necropsied 1.5 hpost injection. The data shows specific targeting of the tumor with[Al¹⁸F]IMP 468 (FIG. 3). Tumor uptake of the peptide is reduced whenbombesin was given 18 min before the Al¹⁸F(IMP468) (FIG. 3).Biodistribution data indicates in vivo stability of Al¹⁸F(IMP468) for atleast 1.5 h (not shown).

Larger tumors showed higher uptake of Al¹⁸F(IMP468), possibly due tohigher receptor expression in larger tumors (not shown). Thebiodistribution data showed Al¹⁸F(IMP468) tumor targeting that was inthe same range as reported for the same peptide labeled with ⁶⁸Ga byPrasanphanich et al. (not shown). The results demonstrate that the ¹⁸Fpeptide labeling method can be used in vivo to target receptors that areupregulated in tumors, using targeting molecules besides antibodies. Inthis case, the IMP468 targeting took advantage of a naturally occurringligand-receptor interaction. The tumor targeting was significant with aP value of P=0.0013. Many such ligand-receptor pairs are known and anysuch targeting interaction may form the basis for ¹⁸F imaging, using themethods described herein.

Example 16 Synthesis and Labeling of Somatostatin Analog IMP466

Somatostatin is another non-antibody targeting peptide that is of usefor imaging the distribution of somatostatin receptor protein.¹²³I-labeled octreotide, a somatostatin analog, has been used forimaging of somatostatin receptor expressing tumors (e.g., Kvols et al.,1993, Radiology 187:129-33; Leitha et al., 1993, J Nucl Med34:1397-1402). However, ¹²³I has not been of extensive use for imagingbecause of its expense, short physical half-life and the difficulty ofpreparing the radiolabeled compounds. The ¹⁸F-labeling methods describedherein are preferred for imaging of somatostatin receptor expressingtumors.

(SEQ ID NO: 50) IMP466 NOTA-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Throl

A NOTA-conjugated derivative of the somatostatin analog octreotide(IMP466) was made by standard Fmoc based solid phase peptide synthesisto produce a linear peptide. The C-terminal Throl residue is threoninol.The peptide was cyclized by treatment with DMSO overnight. The peptide,0.0073 g, 5.59×10⁻⁶ mol was dissolved in 111.9 μL of 0.5 M pH 4 NaOAcbuffer to make a 0.05 M solution of IMP466. The solution formed a gelover time so it was diluted to 0.0125 M by the addition of more 0.5 MNaOAc buffer.

¹⁸F was purified and concentrated with a QMA cartridge to provide 200 μLof ¹⁸F in 0.4 M KHCO₃. The bicarbonate solution was neutralized with 10μL of glacial acetic acid. A 40 μL aliquot of the neutralized ¹⁸F eluentwas mixed with 3 μL of 2 mM AlCl₃, followed by the addition of 40 μL of0.0125 M IMP466 solution. The mixture was heated at 105° C. for 17 min.The reaction was then purified on a Waters 1 cc (30 mg) HLB column byloading the reaction solution onto the column and washing the unbound¹⁸F away with water (3 mL) and then eluting the radiolabeled peptidewith 2×200 μL 1:1 EtOH water. The yield of the radiolabeled peptideafter HLB purification was 34.6%.

Effect of Ionic Strength

To lower the ionic strength of the reaction mixture escalating amountsof acetonitrile were added to the labeling mixture (final concentration:0-80%). The yield of radiolabeled IMP466 increased with increasingconcentration of acetonitrile in the medium. The optimal radiolabelingyield (98%) was obtained in a final concentration of 80% acetonitrile,despite the increased volume (500 μL in 80% vs. 200 μL in 0%acetonitrile). In 0% acetonitrile the radiolabeling yield ranged from36% to 55% in three experiments.

Example 17 Imaging of Neuroendocrine Tumors with an ¹⁸F- and⁶⁸Ga-Labeled IMP466

Studies were performed to compare the PET images obtained using an ¹⁸Fversus ⁶⁸Ga-labeled somatostatin analogue peptide and direct targetingto somatostatin receptor expressing tumors.

Methods

¹⁸F Labeling

IMP466 was synthesized and ¹⁸F-labeled by a variation of the methoddescribed in the Example above. A QMA SEPPAK® light cartridge (Waters,Milford, Mass.) with 2-6 GBq ¹⁸F⁻ (BV Cyclotron VU, Amsterdam, TheNetherlands) was washed with 3 mL metal-free water. ¹⁸F⁻ was eluted fromthe cartridge with 0.4 M KHCO₃ and fractions of 200 μL, were collected.The pH of the fractions was adjusted to pH 4, with 10 μL metal-freeglacial acid. Three μL of 2 mM AlCl₃ in 0.1 M sodium acetate buffer, pH4 were added. Then, 10-50 μL IMP 466 (10 mg/mL) were added in 0.5 Msodium acetate, pH 4.1. The reaction mixture was incubated at 100° C.for 15 min unless stated otherwise. The radiolabeled peptide waspurified on RP-HPLC. The Al¹⁸F(IMP466) containing fractions werecollected and diluted two-fold with H₂O and purified on a 1-cc Oasis HLBcartridge (Waters, Milford, Mass.) to remove acetonitrile and TFA. Inbrief, the fraction was applied on the cartridge and the cartridge waswashed with 3 mL H₂O. The radiolabeled peptide was then eluted with2×200 μL 50% ethanol. For injection in mice, the peptide was dilutedwith 0.9% NaCl. A maximum specific activity of 45,000 GBq/mmol wasobtained.

⁶⁸Ga Labeling

IMP466 was labeled with ⁶⁸GaCl₃ eluted from a TiO₂-based 1,110 MBq⁶⁸Ge/⁶⁸Ga generator (Cyclotron Co. Ltd., Obninsk, Russia) using 0.1 Multrapure HCl (J.T. Baker, Deventer, The Netherlands). IMP466 wasdissolved in 1.0 M HEPES buffer, pH 7.0. Four volumes of ⁶⁸Ga eluate(120-240 MBq) were added and the mixture was heated at 95° C. for 20min. Then 50 mM EDTA was added to a final concentration of 5 mM tocomplex the non-incorporated ⁶⁸Ga³⁺. The ⁶⁸Ga-labeled IMP466 waspurified on an Oasis HLB cartridge and eluted with 50% ethanol.

Octanol-Water Partition Coefficient (Log P_(oct/water))

To determine the lipophilicity of the radiolabeled peptides,approximately 50,000 dpm of the radiolabeled peptide was diluted in 0.5mL phosphate-buffered saline (PBS). An equal volume of octanol was addedto obtain a binary phase system. After vortexing the system for 2 min,the two layers were separated by centrifugation (100×g, 5 min). Three100 μL samples were taken from each layer and radioactivity was measuredin a well-type gamma counter (Wallac Wizard 3″, Perkin-Elmer, Waltham,Mass.).

Stability

Ten μL of the ¹⁸F-labeled IMP466 was incubated in 500 μL of freshlycollected human serum and incubated for 4 h at 37° C. Acetonitrile wasadded and the mixture was vortexed followed by centrifugation at 1000×gfor 5 min to precipitate serum proteins. The supernatant was analyzed onRP-HPLC as described above.

Cell Culture

The AR42J rat pancreatic tumor cell line was cultured in Dulbecco'sModified Eagle's Medium (DMEM) medium (Gibco Life Technologies,Gaithersburg, Md., USA) supplemented with 4500 mg/L D-glucose, 10% (v/v)fetal calf serum, 2 mmol/L glutamine, 100 U/mL penicillin and 100 μg/mLstreptomycin. Cells were cultured at 37° C. in a humidified atmospherewith 5% CO₂.

IC₅₀ Determination

The apparent 50% inhibitory concentration (IC₅₀) for binding thesomatostatin receptors on AR42J cells was determined in a competitivebinding assay using Al¹⁹F(MP466), ⁶⁹Ga(IMP466) or ¹¹⁵In(DTPA-octreotide)to compete for the binding of ¹¹¹In(DTPA-octreotide).

Al¹⁹F(IMP466) was formed by mixing an aluminium fluoride (Al¹⁹F)solution (0.02 M AlCl₃ in 0.5 M NaAc, pH 4, with 0.1 M NaF in 0.5 MNaAc, pH 4) with IMP466 and heating at 100° C. for 15 min. The reactionmixture was purified by RP-HPLC on a C-18 column as described above.

⁶⁹Ga(IMP466) was prepared by dissolving gallium nitrate (2.3×10⁻⁸ mol)in 30 μL mixed with 20 μL IMP466 (1 mg/mL) in 10 mM NaAc, pH 5.5, andheated at 90° C. for 15 min. Samples of the mixture were used withoutfurther purification.

¹¹⁵In(DTPA-octreotide) was made by mixing indium chloride (1×10⁻⁵ mol)with 10 μL DTPA-octreotide (1 mg/mL) in 50 mM NaAc, pH 5.5, andincubated at room temperature (RT) for 15 min. This sample was usedwithout further purification. ¹¹¹In(DTPA-octreotide) (OCTREOSCAN®) wasradiolabeled according to the manufacturer's protocol.

AR42J cells were grown to confluency in 12-well plates and washed twicewith binding buffer (DMEM with 0.5% bovine serum albumin). After 10 minincubation at RT in binding buffer, Al¹⁹F(IMP466), ⁶⁹Ga(IMP466) or¹¹⁵In(DTPA-octreotide) was added at a final concentration ranging from0.1-1000 nM, together with a trace amount (10,000 cpm) of¹¹¹In(DTPA-octreotide) (radiochemical purity >95%). After incubation atRT for 3 h, the cells were washed twice with ice-cold PBS. Cells werescraped and cell-associated radioactivity was determined. Under theseconditions, a limited extent of internalization may occur. We thereforedescribe the results of this competitive binding assay as “apparentIC₅₀” values rather than IC₅₀. The apparent IC₅₀ was defined as thepeptide concentration at which 50% of binding without competitor wasreached.

Biodistribution Studies

Male nude BALB/c mice (6-8 weeks) were injected subcutaneously in theright flank with 0.2 mL AR42J cell suspension of 10⁷ cells/mL.Approximately two weeks after tumor cell inoculation when tumors were5-8 mm in diameter, 370 kBq ¹⁸F or ⁶⁸Ga-labeled IMP466 was administeredintravenously (n=5). Separate groups (n=5) were injected with a1,000-fold molar excess of unlabeled IMP466. One group of three mice wasinjected with unchelated [Al¹⁸F]. All mice were killed by CO₂/O₂asphyxiation 2 h post-injection (p.i.). Organs of interest werecollected, weighed and counted in a gamma counter. The percentage of theinjected dose per gram tissue (% ID/g) was calculated for each tissue.The animal experiments were approved by the local animal welfarecommittee and performed according to national regulations.

PET/CT Imaging

Mice with s.c. AR42J tumors were injected intravenously with 10 MBqAl¹⁸F(IMP466) or ⁶⁸Ga(IMP466). One and two hours after the injection ofpeptide, mice were scanned on an Inveon animal PET/CT scanner (SiemensPreclinical Solutions, Knoxville, Tenn.) with an intrinsic spatialresolution of 1.5 mm (Visser et al, JNM, 2009). The animals were placedin a supine position in the scanner. PET emission scans were acquiredover 15 minutes, followed by a CT scan for anatomical reference (spatialresolution 113 μm, 80 kV, 500 μA). Scans were reconstructed using InveonAcquisition Workplace software version 1.2 (Siemens PreclinicalSolutions, Knoxville, Tenn.) using an ordered set expectationmaximization-3D/maximum a posteriori (OSEM3D/MAP) algorithm with thefollowing parameters: matrix 256×256×159, pixel size 0.43×0.43×0.8 mm³and MAP prior of 0.5 mm.

Results

Effect of Buffer

The effect of the buffer on the labeling efficiency of IMP466 wasinvestigated. IMP466 was dissolved in sodium citrate buffer, sodiumacetate buffer, 2-(N-morpholino)ethanesulfonic acid (MES) or4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at 10mg/mL (7.7 mM). The molarity of all buffers was 1 M and the pH was 4.1.To 200 μg (153 nmol) of IMP466 was added 100 μL, [Al¹⁸F] (pH 4) andincubated at 100° C. for 15 min. Radiolabeling yield and specificactivity was determined with RP-HPLC. When using sodium acetate, MES orHEPES, radiolabeling yield was 49%, 44% and 46%, respectively. In thepresence of sodium citrate, no labeling was observed (<1%). When thelabeling reaction was carried out in sodium acetate buffer, the specificactivity of the preparations was 10,000 GBq/mmol, whereas in MES andHEPES buffer a specific activity of 20,500 and 16,500 GBq/mmol wasobtained, respectively.

Effect of AlCl₃ Concentration

Three stock solutions of AlCl₃ in sodium acetate, pH 4.1 were prepared:0.2, 2.0 and 20 mM. From these solutions, 3 μL was added to 200 μL of¹⁸F⁻ to form [Al¹⁸F]. To these samples, 153 nmol of peptide was addedand incubated for 15 min at 100° C. Radiolabeling yield was 49% afterincubation at a final concentration of 6 nmol AlCl₃. Incubation with 0.6nmol AlCl₃ and 60 nmol AlCl₃ resulted in a strong reduction of theradiolabeling yield: 10% and 6%, respectively.

Effect of Amount of Peptide

The effect of the amount of peptide on the labeling efficiency wasinvestigated. IMP466 was dissolved in sodium acetate buffer, pH 4.1 at aconcentration of 7.7 mM (10 mg/mL) and 38, 153 or 363 nmol of IMP466 wasadded to 200 μL [Al¹⁸F] (581-603 MBq). The radiolabeling yield increasedwith increasing amounts of peptide. At 38 nmol, radiolabeling yieldranged from 4-8%, at 153 nmol, the yield had increased to 42-49% and atthe highest concentration the radiolabeling yield was 48-52%.

Octanol-Water Partition Coefficient

To determine the lipophilicity of the ¹⁸F and ⁶⁸Ga-labeled IMP466, theoctanol-water partition coefficients were determined. The logP_(octanol/water) value for the Al¹⁸F(IMP466) was −2.44±0.12 and that of⁶⁸Ga(IMP466) was −3.79±0.07, indicating that the ¹⁸F-labeled IMP 466 wasslightly less hydrophilic.

Stability

The ¹⁸F-labeled IMP466 did not show release of ¹⁸F after incubation inhuman serum at 37° C. for 4 h, indicating excellent stability of theAl[¹⁸F]NOTA complex.

IC₅₀ Determination

The apparent IC₅₀ of Al¹⁹F(IMP466) was 3.6±0.6 nM, whereas that for⁶⁹Ga(IMP466) was 13±3 nM. The apparent IC₅₀ of the reference peptide,¹¹⁵In(DTPA-octeotride) (OCTREOSCAN®), was 6.3±0.9 nM.

Biodistribution Studies

The biodistribution of both Al¹⁸F(IMP466) and ⁶⁸Ga(IMP466) was studiedin nude BALB/c mice with s.c. AR42J tumors at 2 h p.i. (FIG. 4). Al¹⁸Fwas included as a control. Tumor targeting of the Al¹⁸F(IMP466) was highwith 28.3±5.7% ID/g accumulated at 2 h p.i. Uptake in the presence of anexcess of unlabeled IMP466 was 8.6±0.7% ID/g, indicating that tumoruptake was receptor-mediated. Blood levels were very low (0.10±0.07%ID/g, 2 h pi), resulting in a tumor-to-blood ratio of 299±88. Uptake inthe organs was low, with specific uptake in receptor expressing organssuch as adrenal glands, pancreas and stomach. Bone uptake ofAl¹⁸F(IMP466) was negligible as compared to uptake of non-chelated Al¹⁸F(0.33±0.07 vs. 36.9±5.0% ID/g at 2 h p.i., respectively), indicatinggood in vivo stability of the ¹⁸F-labeled NOTA-peptide.

The biodistribution of Al¹⁸F(IMP466) was compared to that of⁶⁸Ga(IMP466) (FIG. 4). Tumor uptake of ⁶⁸Ga(IMP466) (29.2±0.5% ID/g, 2 hpi) was similar to that of Al¹⁸F-IMP 466 (p<0.001). Lung uptake of⁶⁸Ga(IMP466) was two-fold higher than that of Al¹⁸F(IMP466) (4.0±0.9%ID/g vs. 1.9±0.4% ID/g, respectively). In addition, kidney retention of⁶⁸Ga(IMP466) was slightly higher than that of Al¹⁸F(IMP466) (16.2±2.86%ID/g vs. 9.96±1.27% ID/g, respectively.

Fused PET and CT scans are shown in FIG. 5. PET scans corroborated thebiodistribution data. Both Al¹⁸F(IMP466) and ⁶⁸Ga(IMP466) showed highuptake in the tumor and retention in the kidneys. The activity in thekidneys was mainly localized in the renal cortex. Again, the [Al¹⁸F]proved to be stably chelated by the NOTA chelator, since no bone uptakewas observed.

FIG. 5 clearly shows that the distribution of an ¹⁸F-labeled analog ofsomatostatin (octreotide) mimics that of a ⁶⁸Ga-labeled somatostatinanalog. These results are significant, since ⁶⁸Ga-labeled octreotide PETimaging in human subjects with neuroendocrine tumors has been shown tohave a significantly higher detection rate compared with conventionalsomatostatin receptor scintigraphy and diagnostic CT, with a sensitivityof 97%, a specificity of 92% and an accuracy of 96% (e.g., Gabriel etal., 2007, J Nucl Med 48:508-18). PET imaging with ⁶⁸Ga-labeledoctreotide is reported to be superior to SPECT analysis with¹¹¹In-labeled octreotide and to be highly sensitive for detection ofeven small meningiomas (Henze et al., 2001, J Nucl Med 42:1053-56).Because of the higher energy of ⁶⁸Ga compared with ¹⁸F, it is expectedthat ¹⁸F based PET imaging would show even better spatial resolutionthan ⁶⁸Ga based PET imaging. This is illustrated in FIG. 5 by comparingthe kidney images obtained with ¹⁸F-labeled IMP466 (FIG. 5, left) vs.⁶⁸Ga-labeled IMP466 (FIG. 5, right). The PET images obtained with ⁶⁸Gashow more diffuse margins and lower resolution than the images obtainedwith ¹⁸F. These results demonstrate the superior images obtained with¹⁸F-labeled targeting moieties prepared using the methods andcompositions described herein and confirm the utility of the described¹⁸F-labeling techniques for non-antibody targeting peptides.

Example 18 Comparison of ⁶⁸Ga and ¹⁸F PET Imaging Using Pretargeting

We compared PET images obtained using ⁶⁸Ga- or ¹⁸F-labeled peptides thatwere pretargeted with the bispecific TF2 antibody, prepared as describedabove. The half-lives of ⁶⁸Ga (t_(1/2)=68 minutes) and ¹⁸F (t_(1/2)=110minutes) match with the pharmacokinetics of the radiolabeled peptide,since its maximum accretion in the tumor is reached within hours.Moreover, ⁶⁸Ga is readily available from ⁶⁸Ge/⁶⁸Ga generators, whereas¹⁸F is the most commonly used and widely available radionuclide in PET.

Methods

Mice with s.c. CEA-expressing LS174T tumors received TF2 (6.0 nmol; 0.94mg) and 5 MBq ⁶⁸Ga(IMP288) (0.25 nmol) or Al¹⁸F(IMP449) (0.25 nmol)intravenously, with an interval of 16 hours between the injection of thebispecific antibody and the radiolabeled peptide. One or two hours afterthe injection of the radiolabeled peptide, PET/CT images were acquiredand the biodistribution of the radiolabeled peptide was determined.Uptake in the LS174T tumor was compared with that in an s.c.CEA-negative SK-RC 52 tumor. Pretargeted immunoPET imaging was comparedwith ¹⁸F-FDG PET imaging in mice with an s.c. LS174T tumor andcontralaterally an inflamed thigh muscle.

(SEQ ID NO: 51) IMP288 DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH₂

Pretargeting

The bispecific TF2 antibody was made by the DNL method, as describedabove. TF2 is a trivalent bispecific antibody comprising an HSG-bindingFab fragment from the h679 antibody and two CEA-binding Fab fragmentsfrom the hMN-14 antibody. The DOTA-conjugated, HSG-containing peptideIMP288 was synthesized by peptide synthesis as described above. TheIMP449 peptide, synthesized as described above, contains a1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chelating moiety tofacilitate labeling with ¹⁸F. As a tracer for the antibody component,TF2 was labeled with ¹²⁵I(Perkin Elmer, Waltham, Mass.) by the iodogenmethod (Fraker and Speck, 1978, Biochem Biophys Res Comm 80:849-57), toa specific activity of 58 MBq/nmol.

Labeling of IMP288

IMP288 was labeled with ¹¹¹In (Covidien, Petten, The Netherlands) forbiodistribution studies at a specific activity of 32 MBq/nmol understrict metal-free conditions. IMP288 was labeled with ⁶⁸Ga eluted from aTiO-based 1,110 MBq ⁶⁸Ge/⁶⁸Ga generator (Cyclotron Co. Ltd., ObninskRussia) using 0.1 M ultrapure HCl. Five 1 ml fractions were collectedand the second fraction was used for labeling the peptide. One volume of1.0 M HEPES buffer, pH 7.0 was added to 3.4 nmol IMP 288. Four volumesof ⁶⁸Ga eluate (380 MBq) were added and the mixture was heated to 95° C.for 20 min. Then 50 mM EDTA was added to a final concentration of 5 mMto complex the non-chelated ⁶⁸Ga³⁺. The ⁶⁸Ga(IMP288) peptide waspurified on a 1-mL Oasis HLB-cartridge (Waters, Milford, Mass.). Afterwashing the cartridge with water, the peptide was eluted with 25%ethanol. The procedure to label IMP288 with ⁶⁸Ga was performed within 45minutes, with the preparations being ready for in vivo use.

Labeling of IMP449

IMP449 was labeled with ¹⁸F as described above. 555-740 MBq ¹⁸F (B.V.Cyclotron VU, Amsterdam, The Netherlands) was eluted from a QMAcartridge with 0.4 M KHCO₃. The Al¹⁸F activity was added to a vialcontaining the peptide (230 μg) and ascorbic acid (10 mg). The mixturewas incubated at 100° C. for 15 min. The reaction mixture was purifiedby RP-HPLC. After adding one volume of water, the peptide was purifiedon a 1-mL Oasis HLB cartridge. After washing with water, theradiolabeled peptide was eluted with 50% ethanol. Al¹⁸F(IMP449) wasprepared within 60 minutes, with the preparations being ready for invivo use.

Radiochemical purity of ¹²⁵I-TF2, ¹¹¹In(IMP288) and ⁶⁸Ga(IMP288) andAl¹⁸F(IMP449) preparations used in the studies always exceeded 95%.

Animal Experiments

Experiments were performed in male nude BALB/c mice (6-8 weeks old),weighing 20-25 grams. Mice received a subcutaneous injection with 0.2 mLof a suspension of 1×10⁶ LS174T-cells, a CEA-expressing human coloncarcinoma cell line (American Type Culture Collection, Rockville, Md.,USA). Studies were initiated when the tumors reached a size of about0.1-0.3 g (10-14 days after tumor inoculation).

The interval between TF2 and IMP288 injection was 16 hours, as thisperiod was sufficient to clear unbound TF2 from the circulation. In somestudies ¹²⁵I-TF2, (0.4 MBq) was co-injected with unlabeled TF2. IMP288was labeled with either ¹¹¹In or ⁶⁸Ga. IMP449 was labeled with ¹⁸F. Micereceived TF2 and IMP288 intravenously (0.2 mL). One hour after theinjection of ⁶⁸Ga-labeled peptide, and two hours after injection ofAl¹⁸F(IMP449), mice were euthanized by CO₂/O₂, and blood was obtained bycardiac puncture and tissues were dissected.

PET images were acquired with an Inveon animal PET/CT scanner (SiemensPreclinical Solutions, Knoxville, Tenn.). PET emission scans wereacquired for 15 minutes, preceded by CT scans for anatomical reference(spatial resolution 113 μm, 80 kV, 500 μA, exposure time 300 msec).

After imaging, tumor and organs of interest were dissected, weighed andcounted in a gamma counter with appropriate energy windows for ¹²⁵I,¹¹¹In, ⁶⁸Ga or ¹⁸F. The percentage-injected dose per gram tissue (%ID/g) was calculated.

Results

Within 1 hour, pretargeted immunoPET resulted in high and specifictargeting of ⁶⁸Ga-IMP288 in the tumor (10.7±3.6% ID/g), with very lowuptake in the normal tissues (e.g., tumor/blood 69.9±32.3), in aCEA-negative tumor (0.35±0.35% ID/g), and inflamed muscle (0.72±0.20%ID/g). Tumors that were not pretargeted with TF2 also had low⁶⁸Ga(IMP288) uptake (0.20±0.03% ID/g). [¹⁸F]FDG accreted efficiently inthe tumor (7.42±0.20% ID/g), but also in the inflamed muscle (4.07±1.13%ID/g) and a number of normal tissues, and thus pretargeted ⁶⁸Ga-IMP 288provided better specificity and sensitivity. The corresponding PET/CTimages of mice that received ⁶⁸Ga(IMP288) or Al¹⁸F(IMP449) followingpretargeting with TF2 clearly showed the efficient targeting of theradiolabeled peptide in the subcutaneous LS174T tumor, while theinflamed muscle was not visualized. In contrast, with ¹⁸F-FDG the tumoras well as the inflammation was clearly delineated.

Dose Optimization

The effect of the TF2 bsMAb dose on tumor targeting of a fixed 0.01 nmol(15 ng) dose of IMP288 was determined. Groups of five mice were injectedintravenously with 0.10, 0.25, 0.50 or 1.0 nmol TF2 (16, 40, 80 or 160μg respectively), labeled with a trace amount of ¹²⁵I (0.4 MBq). Onehour after injection of ¹¹¹In(IMP288) (0.01 nmol, 0.4 MBq), thebiodistribution of the radiolabels was determined.

TF2 cleared rapidly from the blood and the normal tissues. Eighteenhours after injection the blood concentration was less than 0.45% ID/gat all TF2 doses tested. Targeting of TF2 in the tumor was 3.5% ID/g at2 h p.i. and independent of TF2 dose (data not shown). At all TF2 doses¹¹¹In(IMP288) accumulated effectively in the tumor (not shown). Athigher TF2 doses enhanced uptake of ¹¹¹In(IMP288) in the tumor wasobserved: at 1.0 nmol TF2 dose maximum targeting of IMP288 was reached(26.2±3.8% ID/g). Thus at the 0.01 nmol peptide dose highest tumortargeting and tumor-to-blood ratios were reached at the highest TF2 doseof 1.0 nmol (TF2:IMP288 molar ratio 100:1). Among the normal tissues,the kidneys had the highest uptake of ¹¹¹In(IMP288) (1.75±0.27% ID/g)and uptake in the kidneys was not affected by the TF2 dose (not shown).All other normal tissues had very low uptake, resulting in extremelyhigh tumor-to-nontumor ratios, exceeding 50:1 at all TF2 doses tested(not shown).

For PET imaging using ⁶⁸Ga-labeled IMP288, a higher peptide dose isrequired, because a minimum activity of 5-10 MBq ⁶⁸Ga needs to beinjected per mouse if PET imaging is performed 1 h after injection. Thespecific activity of the ⁶⁸Ga(IMP288) preparations was 50-125 MBq/nmolat the time of injection. Therefore, for PET imaging at least 0.1-0.25nmol of IMP288 had to be administered. The same TF2:IMP288 molar ratioswere tested at 0.1 nmol IMP288 dose. LS174T tumors were pretargeted byinjecting 1.0, 2.5, 5.0 or 10.0 nmol TF2 (160, 400, 800 or 1600 μg). Incontrast to the results at the lower peptide dose, ¹¹¹In(IMP288) uptakein the tumor was not affected by the TF2 doses (15% ID/g at all dosestested, data not shown). TF2 targeting in the tumor in terms of % Digdecreased at higher doses (3.21±0.61% ID/g versus 1.16±0.27% ID/g at aninjected dose of 1.0 nmol and 10.0 nmol, respectively) (data not shown).Kidney uptake was also independent of the bsMAb dose (2% ID/g). Based onthese data we selected a bsMAb dose of 6.0 nmol for targeting 0.1-0.25nmol of IMP288 to the tumor.

PET Imaging

To demonstrate the effectiveness of pretargeted immunoPET imaging withTF2 and ⁶⁸Ga(IMP288) to image CEA-expressing tumors, subcutaneous tumorswere induced in five mice. In the right flank an s.c. LS 174T tumor wasinduced, while at the same time in the same mice 1×10⁶ SK-RC 52 cellswere inoculated in the left flank to induce a CEA-negative tumor.Fourteen days later, when tumors had a size of 0.1-0.2 g, the mice werepretargeted with 6.0 nmol ¹²⁵I-TF2 intravenously. After 16 hours themice received 5 MBq ⁶⁸Ga(IMP288) (0.25 nmol, specific activity of 20MBq/nmol). A separate group of three mice received the same amount of⁶⁸Ga-IMP 288 alone, without pretargeting with TF2. PET/CT scans of themice were acquired 1 h after injection of the ⁶⁸Ga(IMP288).

The biodistribution of ¹²⁵I-TF2 and [⁶⁸Ga]IMP288 in the mice are shownin FIG. 6. Again high uptake of the bsMAb (2.17±0.50% ID/g) and peptide(10.7±3.6% ID/g) in the tumor was observed, with very low uptake in thenormal tissues (tumor-to-blood ratio: 64±22). Targeting of ⁶⁸Ga(IMP288)in the CEA-negative tumor SK-RC 52 was very low (0.35±0.35% ID/g).Likewise, tumors that were not pretargeted with TF2 had low uptake of⁶⁸Ga(IMP288) (0.20±0.03% ID/g), indicating the specific accumulation ofIMP288 in the CEA-expressing LS 174T tumor.

The specific uptake of ⁶⁸Ga(IMP288) in the CEA-expressing tumorpretargeted with TF2 was clearly visualized in a PET image acquired 1 hafter injection of the ⁶⁸Ga-labeled peptide (not shown). Uptake in thetumor was evaluated quantitatively by drawing regions of interest (ROI),using a 50% threshold of maximum intensity. A region in the abdomen wasused as background region. The tumor-to-background ratio in the image ofthe mouse that received TF2 and ⁶⁸Ga(IMP288) was 38.2.

We then examined pretargeted immunoPET with ¹⁸F-FDG. In two groups offive mice a s.c. LS174T tumor was induced on the right hind leg and aninflammatory focus in the left thigh muscle was induced by intramuscularinjection of 50 μL turpentine (18). Three days after injection of theturpentine, one group of mice received 6.0 nmol TF2, followed 16 h laterby 5 MBq ⁶⁸Ga(IMP288) (0.25 nmol). The other group received ¹⁸F-FDG (5MBq). Mice were fasted during 10 hours prior to the injection andanaesthetized and kept warm at 37° C. until euthanasia, 1 hpostinjection.

Uptake of ⁶⁸Ga(IMP288) in the inflamed muscle was very low, while uptakein the tumor in the same animal was high (0.72±0.20% ID/g versus8.73±1.60% ID/g, p<0.05, FIG. 7). Uptake in the inflamed muscle was inthe same range as uptake in the lungs, liver and spleen (0.50±0.14%ID/g, 0.72±0.07% ID/g, 0.44±0.10% ID/g, respectively). Tumor-to-bloodratio of ⁶⁸Ga(IMP288) in these mice was 69.9±32.3; inflamedmuscle-to-blood ratio was 5.9±2.9; tumor-to-inflamed muscle ratio was12.5±2.1. In the other group of mice ¹⁸F-FDG accreted efficiently in thetumor (7.42±0.20% ID/g, tumor-to-blood ratio 6.24±1.5, FIG. 4). ¹⁸F-FDGalso substantially accumulated in the inflamed muscle (4.07±1.13% ID/g),with inflamed muscle-to-blood ratio of 3.4±0.5, and tumor-to-inflamedmuscle ratio of 1.97±0.71.

The corresponding PET/CT image of a mouse that received ⁶⁸Ga(IMP288),following pretargeting with TF2, clearly showed the efficient accretionof the radiolabeled peptide in the tumor, while the inflamed muscle wasnot visualized (FIG. 8). In contrast, on the images of the mice thatreceived ¹⁸F-FDG, the tumor as well as the inflammation was visible(FIG. 8). In the mice that received ⁶⁸Ga(IMP288), the tumor-to-inflamedtissue ratio was 5.4; tumor-to-background ratio was 48; inflamedmuscle-to-background ratio was 8.9. ¹⁸F-FDG uptake had atumor-to-inflamed muscle ratio of 0.83; tumor-to-background ratio was2.4; inflamed muscle-to-background ratio was 2.9.

The pretargeted immunoPET imaging method was tested using theAl¹⁸F(IMP449). Five mice received 6.0 nmol TF2, followed 16 h later by 5MBq Al[¹⁸F]IMP449 (0.25 nmol). Three additional mice received 5 MBqAl¹⁸F(IMP449) without prior administration of TF2, while two controlmice were injected with [Al¹⁸F] (3 MBq). The results of this experimentare summarized in FIG. 9. Uptake of Al¹⁸F(IMP449) in tumors pretargetedwith TF2 was high (10.6±1.7% ID/g), whereas it was very low in thenon-pretargeted mice (0.45±0.38% ID/g). [Al¹⁸F] accumulated in the bone(50.9±11.4% ID/g), while uptake of the radiolabeled IMP449 peptide inthe bone was very low (0.54±0.2% ID/g), indicating that theAl¹⁸F(IMP449) was stable in vivo. The biodistribution of Al¹⁸F(IMP449)in the TF2 pretargeted mice with s.c. LS174T tumors were highly similarto that of ⁶⁸Ga(IMP288).

The PET-images of pretargeted immunoPET with Al¹⁸F(IMP449) show the sameintensity in the tumor as those with ⁶⁸Ga(IMP288), but the resolution ofthe ¹⁸F PET images were superior to those of the ⁶⁸Ga. (FIG. 10). Thetumor-to-background ratio of the Al¹⁸F(IMP449) signal was 66.

Conclusions

The present study showed that pretargeted immunoPET with theanti-CEA×anti-HSG bispecific antibody TF2 in combination with a ⁶⁸Ga- or¹⁸F-labeled di-HSG-DOTA-peptide is a rapid and specific technique forPET imaging of CEA-expressing tumors.

Pretargeted immunoPET with TF2 in combination with ⁶⁸Ga(IMP288) orAl¹⁸F(IMP449) involves two intravenous administrations. An intervalbetween the infusion of the bsMAb and the radiolabeled peptide of 16 hwas used. After 16 h most of the TF2 had cleared from the blood (bloodconcentration <1% ID/g), preventing complexation of TF2 and IMP288 inthe circulation.

For these studies the procedure to label IMP288 with ⁶⁸Ga was optimized,resulting in a one-step labeling technique. We found that purificationon a C18/HLB cartridge was needed to remove the ⁶⁸Ga colloid that isformed when the peptide was labeled at specific activities exceeding 150GBq/nmol at 95° C. If a preparation contains a small percentage ofcolloid and is administered intravenously, the ⁶⁸Ga colloid accumulatesin tissues of the mononuclear phagocyte system (liver, spleen, and bonemarrow), deteriorating image quality. The ⁶⁸Ga-labeled peptide could berapidly purified on a C18-cartridge. Radiolabeling and purification foradministration could be accomplished within 45 minutes.

The half-life of ⁶⁸Ga matches with the kinetics of the IMP288 peptide inthe pretargeting system: maximum accretion in the tumor is reachedwithin 1 h. ⁶⁸Ga can be eluted twice a day form a ⁶⁸Ge/⁶⁸Ga generator,avoiding the need for an on-site cyclotron. However, the high energy ofthe positrons emitted by ⁶⁸Ga (1.9 MeV) limits the spatial resolution ofthe acquired images to 3 mm, while the intrinsic resolution of themicroPET system is as low as 1.5 mm.

¹⁸F, the most widely used radionuclide in PET, has an even morefavorable half-life for pretargeted PET imaging (t_(1/2)=110 min). TheNOTA-conjugated peptide IMP449 was labeled with ¹⁸F, as described above.Like labeling with ⁶⁸Ga, it is a one-step procedure. Labeling yields ashigh as 50% were obtained. The biodistribution of Al¹⁸F(IMP449) washighly similar to that of ⁶⁸Ga-labeled IMP288, suggesting that with thislabeling method ¹⁸F is a residualizing radionuclide.

In contrast with FDG-PET, pretargeted radioimmunodetection is a tumorspecific imaging modality. Although a high sensitivity and specificityfor FDG-PET in detecting recurrent colorectal cancer lesions has beenreported in patients (Huebner et al., 2000, J Nucl Med 41:11277-89),FDG-PET images could lead to diagnostic dilemmas in discriminatingmalignant from benign lesions, as indicated by the high level oflabeling observed with inflammation. In contrast, the hightumor-to-background ratio and clear visualization of CEA-positive tumorsusing pretargeted immunoPET with TF2 ⁶⁸Ga- or ¹⁸F-labeled peptidessupports the use of the described methods for clinical imaging of cancerand other conditions. Apart from detecting metastases and discriminatingCEA-positive tumors from other lesions, pretargeted immunoPET could alsobe used to estimate radiation dose delivery to tumor and normal tissuesprior to pretargeted radioimmunotherapy. As TF2 is a humanized antibody,it has a low immunogenicity, opening the way for multiple imaging ortreatment cycles.

Example 19 Synthesis of Folic Acid NOTA Conjugate

Folic acid is activated as described (Wang et. al. Bioconjugate Chem.1996, 7, 56-62.) and conjugated to Boc-NH—CH₂—CH₂—NH₂. The conjugate ispurified by chromatography. The Boc group is then removed by treatmentwith TFA. The amino folate derivative is then mixed with p-SCN-Bn-NOTA(Macrocyclics) in a carbonate buffer. The product is then purified byHPLC. The folate-NOTA derivative is labeled with Al¹⁸F as described inthe preceding Examples and then HPLC purified. The ¹⁸F-labeled folate isinjected i.v. into a subject and successfully used to image thedistribution of folate receptors, for example in cancer or inflammatorydiseases (see, e.g., Ke et al., Advanced Drug Delivery Reviews,56:1143-60, 2004).

Example 20 Pretargeted PET Imaging in Humans

A patient (1.7 m² body surface area) with a suspected recurrent tumor isinjected with 17 mg of bispecific monoclonal antibody (bsMab). The bsMabis allowed to localize to the target and clear from the blood. The¹⁸F-labeled peptide (5-10 mCi on 5.7×10⁻⁹ mol) is injected when 99% ofthe bsMab has cleared from the blood. PET imaging shows the presence ofmicrometastatic tumors.

Example 21 Imaging of Angiogenesis Receptors by ¹⁸F-Labeling

Labeled Arg-Gly-Asp (RGD) peptides have been used for imaging ofangiogenesis, for example in ischemic tissues, where α_(v)β₃ integrin isinvolved. (Jeong et al., J. Nucl. Med. 2008, Apr. 15 epub). RGD isconjugated to SCN-Bn-NOTA according to Jeong et al. (2008). [Al¹⁸F] isattached to the NOTA-derivatized RGD peptide as described above, bymixing aluminum stock solution with ¹⁸F and the derivatized RGD peptideand heating at 110° C. for 15 min, using an excess of peptide to drivethe labeling reaction towards completion. The ¹⁸F labeled RGD peptide isused for in vivo biodistribution and PET imaging as disclosed in Jeonget al. (2008). The [Al¹⁸F] conjugate of RGD-NOTA is taken up intoischemic tissues and provides PET imaging of angiogenesis.

Example 22 Carbohydrate Labeling

A NOTA thiosemicarbazide derivative is prepared by reacting thep-SCN-Bn-NOTA with hydrazine and then purifying the ligand by HPLC.[Al¹⁸F] is prepared as described in the preceding Examples and the[Al¹⁸F] is added to the NOTA thiosemicarbazide and heated for 15 min.Optionally the Al¹⁸F(NOTA thiosemicarbazide) complex is purified byHPLC. The Al¹⁸F(NOTA thiosemicarbazide) is conjugated to oxidizedcarbohydrates by known methods. The ¹⁸F-labeled carbohydrate issuccessfully used for imaging studies using PET scanning.

Example 23 Effect of Organic Solvents on F-18 Labeling

The affinity of chelating moieties such as NETA and NOTA for aluminum ismuch higher than the affinity of aluminum for ¹⁸F. The affinity of Alfor ¹⁸F is affected by factors such as the ionic strength of thesolution, since the presence of other counter-ions tends to shield thepositively charged aluminum and negatively charged fluoride ions fromeach other and therefore to decrease the strength of ionic binding.Therefore low ionic strength medium should increase the effectivebinding of Al and ¹⁸F.

An initial study adding ethanol to the ¹⁸F reaction was found toincrease the yield of radiolabeled peptide. IMP461 was prepared asdescribed above.

TABLE 9 ¹⁸F-labeling of IMP461 in ethanol # 2 mM AlCl₃ ¹⁸F 2 mM IMP 461Solvent Yield* 1 10 μL 741 μCi 20 μL EtOH 60 μL 64.9% 2 10 μL 739 μCi 20μL H₂O 60 μL 21.4% 3 10 μL 747 μCi 20 μL EtOH 60 μL 46.7% 4  5 μL 947μCi 10 μL EtOH 60 μL 43.2% *Yield after HLB column purification, Rxn #1, 2 and 4 were heated to 101° C. for 5 minutes, Rxn # 3 was heated for1 minute in a microwave oven.

Preliminary results showed that addition of ethanol to the reactionmixture more than doubled the yield of ¹⁸F-labeled peptide. Table 9 alsoshows that microwave irradiation can be used in place of heating topromote incorporation of [Al¹⁸F] into the chelating moiety of IMP461.Sixty seconds of microwave radiation (#3) appeared to be slightly less(18%) effective than heating to 101° C. for 5 minutes (#1).

The effect of additional solvents on Al¹⁹F complexation of peptides wasexamined. In each case, the concentration of reactants was the same andonly the solvent varied. Reaction conditions included mixing 25 μLNa¹⁹F+20 μL AlCl₃+20 μL IMP461+60 μL solvent, followed by heating at101° C. for 5 min. Table 10 shows that the presence of a solvent doesimprove the yields of Al¹⁹F(IMP461) (i.e., IMP473) considerably.

TABLE 10 Complexation of IMP 461 with Al¹⁹F in various solvents SolventH₂O MeOH EtOH CH₃CN Al-IMP461 2.97 3.03 2.13 1.54 IMP465 52.46 34.1931.58 24.58 IMP473 14.99 30.96 33.00 37.48 IMP473 15.96 31.81 33.2936.40 IMP461 13.63 — — — Solvent IPA Acetone THF Dioxane Al-IMP461 2.022.05 2.20 16.67 IMP465 32.11 28.47 34.76 10.35 IMP473 27.31 34.35 29.3827.09 IMP473 27.97 35.13 29.28 11.62 IMP461 10.58 — 4.37 34.27 SolventDMF DMSO t_(R) (min) Al-IMP461 — — 9.739 IMP465 19.97 37.03 10.138IMP473 27.77 31.67 11.729 IMP473 27.34 31.29 11.952 IMP461 — — 12.535Al[¹⁹F]IMP461 = IMP473

Example 24 Elution of ¹⁸F⁻ with Bicarbonate

¹⁸F, 10.43 mCi, was received in 2 mL in a syringe. The solution waspassed through a SEP-PAK® Light, WATERS® ACCELL™ Plus QMA Cartridge. Thecolumn was then washed with 5 mL of DI water. The ¹⁸F was eluted with0.4 M KHCO₃ in fractions as shown in Table 11 below.

TABLE 11 Elution of QMA Cartridge with KHCO₃ Vol. Acetic Vol. 0.4M Vialacid μL KHCO₃ μL Activity mCi 1 7.5 150 0.0208 2 10 200 7.06 3 5 1001.653 4 25 500 0.548

The effects of the amount of additional solvent (CH₃CN) on ¹⁸F-labelingof IMP461 was examined. In each case, the concentration of reactants wasthe same and only the amount of solvent varied. Reaction conditionsincluded mixing 10 μL AlCl₃+20 μL ¹⁸F+20 μL IMP461+CH₃CN followed byheating at 101° C. for 5 min. Table 12 shows that following an initialimprovement the labeling efficiency decreases in the presence of excesssolvent.

TABLE 12 ¹⁸F-labeling of IMP461 using varying amounts of CH₃CN CH₃CNt_(R) 2.70 min t_(R) 8.70 min RCY % (μL) ¹⁸F⁻ mCi (%) (%) (HLB) 0 0.64213.48 86.52 50.7 100 0.645 1.55 98.45 81.8* 200 0.642 2.85 97.15 80.8400 0.645 14.51 85.49 57.8 *Aqueous wash contains labeled peptide. RCY =radiochemical yield after HLB purification

Example 25 High Dose Radiolabeling of IMP461

¹⁸F⁻, 163 mCi, was received in 2 mL in a syringe. The solution waspassed through a SEP-PAK® Light, WATERS® ACCELL™ Plus QMA Cartridge. Thecolumn was then washed with 5 mL of DI water. The ¹⁸F⁻ was eluted with0.4 M K₂CO₃ in fractions as shown in Table 13.

TABLE 13 High Dose Labeling Vol. Acetic Vol. 0.4M Vial acid μL K₂CO₃ μLActivity mCi 1 18.5 185 5.59 2 5 50 35.8 3 5 50 59.9 4 5 50 20.5 5 5 505.58 6 50 500 4.21

An aluminum chloride solution (10 μL, 2 mM in pH 4, 2 mM NaOAc) wasadded to vial number 3 from Table 13. The peptide (20 μL, 2 mM in pH 4,2 mM NaOAc) was added to the vial followed by the addition of 170 μL ofCH₃CN. The solution was heated for 10 min at 103° C. the diluted with 6mL of water. The solution was pulled into a 10 mL syringe and injectedonto two WATERS® HLB Plus Cartridges arranged in tandem. The cartridgeswere washed with 8 mL water. The radiolabeled peptide Al¹⁸F(IMP461) wasthen eluted with 10 mL 1:1 EtOH/H₂O, 30.3 mCi, 63.5% yield, specificactivity 750 Ci/mmol. The labeled peptide was free of unbound ¹⁸F byHPLC. The total reaction and purification time was 20 min.

Example 26 Preparation of Al¹⁹F Peptides

Products containing ²⁷Al and/or ¹⁹F are useful for certain applicationslike MR imaging. An improved method for preparing [Al¹⁹F] compounds wasdeveloped. IMP461 was prepared as described above and labeled with ¹⁹F.Reacting IMP461 with AlCl₃+NaF resulted in the formation of threeproducts (not shown). However, by reacting IMP461 with AlF₃.3H₂O weobtained a higher yield of Al¹⁹F(IMP461).

Synthesis of IMP 473

[Al¹⁹F(IMP461)] To (14.1 mg, 10.90 μmol) IMP461 in 2 mL NaOAc (2 mM, pH4.18) solution added (4.51 mg, 32.68 μmol) AlF₃.3H₂O and 500 μL ethanol.The pH of the solution to adjusted to 4.46 using 3 μL 1 N NaOH andheated in a boiling water bath for 30 minutes. The crude reactionmixture was purified by preparative RP-HPLC to yield 4.8 mg (32.9%) ofIMP 473. HRMS (ESI-TOF) MH⁺ expected 1337.6341; found 1337.6332

These results demonstrate that ¹⁹F labeled molecules may be prepared byforming metal-¹⁹F complexes and binding the metal-¹⁹F to a chelatingmoiety, as discussed above for ¹⁸F labeling. The instant Example showsthat a targeting peptide of use for pretargeting detection, diagnosisand/or imaging may be prepared using the instant methods.

Example 27 Synthesis and Labeling of IMP479, IMP485 and IMP487

The structures of additional peptides (IMP479, IMP485, and IMP487)designed for ¹⁸F-labeling are shown in FIG. 11 to FIG. 13. IMP485 isshown in FIG. 12. IMP485 was made on Sieber Amide resin by adding thefollowing amino acids to the resin in the order shown:Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc was cleaved,Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc wascleaved, (tert-Butyl)₂NODA-MPAA (methyl phenyl acetic acid). The peptidewas then cleaved from the resin and purified by RP-HPLC to yield 44.8 mgof IMP485.

Synthesis of Bis-t-butyl-NODA-MPAA (tBu)₂NODA-MPAA for IMP485 Synthesis

To a solution of 4-(bromomethyl) phenyl acetic acid (Aldrich 310417)(0.5925 g, 2.59 mmol) in CH₃CN (anhydrous) (50 mL) at 0° C. was addeddropwise over 1 h a solution of NO2AtBu (1.0087 g, 2.82 mmol) in CH₃CN(50 mL). After 4 h anhydrous K₂CO₃ (0.1008 g, 0.729 mmol) was added tothe reaction mixture and allowed to stir at room temperature overnight.Solvent was evaporated and the crude mixture was purified by preparativeRP-HPLC to yield a white solid (0.7132 g, 54.5%).

Although this is a one step synthesis, yields were low due toesterification of the product by 4-(bromomethyl)phenylacetic acid.Alkylation of NO2AtBu using methyl(4-bromomethyl)phenylacetate wasemployed to prevent esterification (FIG. 14).

¹⁸F-Labeling

For ¹⁸F labeling studies in water, to 40 nmol of IMP479/485/487(formulated using trehalose+ascorbic acid+AlCl₃) was added 250 μL ¹⁸F⁻solution [˜919-1112 μCi of ¹⁸F⁻] and heated to 101° C. for 15 minutes.In ethanol, to 40 nmol of IMP479/485/487 (formulated usingtrehalose+ascorbic acid+AlCl₃) was added 250 μL ¹⁸F⁻ solution[1.248-1.693 mCi of ¹⁸F⁻], 100 μL EtOH and heated to 101° C. for 15minutes. An exemplary experiment showing labeling of different peptidesis shown in Table 14. With minimal optimization, radiolabeling of IMP485has been observed with up to an 88% yield and a specific activity of2,500 Ci/mmol. At this specific activity, HPLC purification of theradiolabeled peptide is not required for in vivo PET imaging using theradiolabeled peptide.

TABLE 14 Labeling of IMP479, IMP485 and IMP487 Isolated yields after HLBpurification IMP # H₂O EtOH IMP479 44.0% 57.5% IMP485 74.4% 79.7% IMP48763.6% 81.6%

Stability in Serum

A kit containing 40 nmol of IMP485 or IMP487, 20 nmol AlCl₃, 0.1 mgascorbic acid and 0.1 g trehalose adjusted to pH 3.9 was reconstitutedwith purified ¹⁸F⁻ in 200 μL saline and heated 106° C. for 15 min. Thereaction mixture was then diluted with 800 μL water and placed on an HLBcolumn. After washing, the column was eluted with 2×200 μL 1:1 EtOH/H₂Oto obtain the purified Al¹⁸F(IMP485) in 64.6% isolated yield. Theradiolabeled peptide in 50 μL was mixed with 250 μL of fresh human serumin a vial and incubated at 37° C.

Both radiolabeled peptides were stable at 37° C. in fresh human serumover the four hours tested (not shown).

Effect of Bulking Agents on Yield of Lyophilized Peptide

An experiment was performed to compare yield using IMP485 kits (40 nmol)with different bulking agents labeled with 2 mCi of F-18 (from the samebatch of F-18) in 200 microliters of saline. The bulking agents wereintroduced at a concentration of 5% by weight in water with a dose of200 microliters/vial. We tested sorbitol, trehalose, sucrose, mannitoland glycine as bulking agents. Results are shown in Table 15

TABLE 15 Effects of Bulking Agents on Radiolabeling Yield Bulking AgentActivity mCi Yield % Sorbitol 2.17 82.9 Glycine 2.17 41.5 Mannitol 2.1181.8 Sucrose 2.11 66.1 Trehalose 2.10 81.3

Sorbitol, mannitol and trehalose all gave radiolabeled product in thesame yield. The mannitol kit and the trehalose kit both formed nicecakes. The sucrose kit and the glycine kit both had significantly loweryields. We also recently tested 2-hydroxypropyl-beta-cyclodextrin as abulking agent and obtained a 58% yield for the 40 nmol kit. We havefound that radiolabeling is very pH sensitive and needs to be tuned tothe ligand and possibly even to the peptide+the ligand. In the case ofIMP485 the optimal pH is pH 4.0±0.2 whereas the optimal pH for IMP467was pH 4.5±0.5. In both cases the yields drop off rapidly outside theideal pH zone.

Time Course of Labeling

The time course for labeling of IMP485 was examined. To 40 nmol ofIMP485 (formulated using trehalose+AlCl₃ (20 nmol)+ascorbic acid) wasadded ˜200-250 μL ¹⁸F⁻ solution (0.9% saline) and heated to 104° C. for5 to 15 minutes. The results for labeling yield were: 5 min (28.9%), 10min (57.9%), 15 min (83.7%) and 30 min (88.9%). Thus, the time coursefor labeling was approximately 15 minutes.

Biodistribution of IMP485 Alone

The biodistribution of IMP485 in the absence of any pretargetingantibody was examined in female Taconic nude mice (10 week old) bearingsmall or no BXPC3 pancreatic cancer xenografts. The mice were injectedi.v. with Al¹⁸F(IMP485), (340 μCi, 2.29×10⁻⁹ mol, 100 μL in saline). Themice, 6 per time point, were necropsied at 30 min and 90 min postinjection. In the absence of pretargeting antibody a low level ofaccumulation was seen in tumor and most normal tissues. The substantialmajority of radiolabel was found in the bladder and to a lesser extentin kidney. Most of the activity was cleared before the 90 min timepoint.

Pretargeting of IMP485 with TF2 DNL Targeting Molecule

IMP485 Radiolabeling

¹⁸F⁻ (218 mCi) was purified to isolate 145.9 mCi. The purified ¹⁸F⁻ (135mCi) was added to a lyophilized vial containing 40 nmol of pre-complexedAl(IMP485). The reaction vial was heated at 110° C. for 17 min. Water(0.8 mL) was added to the reaction mixture before HLB purification. Theproduct (22 mCi) was eluted with 0.6 mL of water:ethanol (1:1) mixtureinto a vial containing lyophilized ascorbic acid. The product wasdiluted with saline. The Al¹⁸F(IMP485) specific activity used forinjection was 550 Ci/mmol.

Biodistribution of Al¹⁸F(IMP485) Alone

Mice bearing sc LS174T xenografts were injected with Al¹⁸F(IMP485) (28μCi, 5.2×10⁻¹¹ mol, 100 μL. Mice were necropsied at 1 and 3 h postinjection, 6 mice per time point.

Biodistribution of TF2+Al¹⁸F(IMP485) with Pretargeting at 20:1 bsMAb toPeptide Ratio

Mice bearing sc LS174T xenografts were injected with TF2 (163.2 μg,1.03×10⁻⁹ mol, iv) and allowed 16.3 h for clearance before injectingAl¹⁸F(IMP485) (28 μCi, 5.2×10⁻¹¹ mol, 100 μL iv). Mice were necropsiedat 1 and 3 h post injection, 7 mice per time point.

Urine Stability

Ten mice bearing s.c. Capan-1 xenografts were injected withAl¹⁸F(IMP485) (400 μCi, in saline, 100 μL). Urine was collected from 3mice at 55 min post injection. The urine samples were analyzed byreverse phase and SE-HPLC. Stability of the radiolabeled IMP485 in urinewas observed.

TABLE 16 Al¹⁸F(IMP485) Alone at 1 h post injection: Tissue n Weight STDWT % ID/g STD % ID/g % ID/org STD % ID/org T/NT STD T/NT Tumor 6 0.2350.147 0.316 0.114 0.081 0.063 1.0 0.0 Liver 6 1.251 0.139 0.176 0.0320.220 0.043 1.8 0.4 Spleen 6 0.085 0.019 0.210 0.181 0.018 0.017 1.9 0.9Kidney 6 0.149 0.013 3.328 0.556 0.499 0.119 0.1 0.0 Lung 6 0.141 0.0390.238 0.048 0.033 0.010 1.3 0.3 Blood 6 0.222 0.006 0.165 0.062 0.2680.101 2.0 0.4 Stomach 6 0.478 0.083 0.126 0.110 0.057 0.045 3.5 1.6 SmInt. 6 0.896 0.098 0.396 0.128 0.353 0.110 0.8 0.3 Lg Int. 6 0.504 0.0560.081 0.019 0.041 0.010 3.9 0.9 Muscle 6 0.103 0.029 0.114 0.079 0.0110.008 4.1 2.5 Scapula 6 0.057 0.015 0.107 0.019 0.006 0.001 2.9 0.7

TABLE 17 Al¹⁸F(IMP485) Alone at 3 h post injection: STD STD STD STDTissue n Weight WT % ID/g % ID/g % ID/org % ID/org T/NT T/NT Tumor 60.265 0.126 0.088 0.020 0.022 0.011 1.0 0.0 Liver 6 1.219 0.091 0.0950.047 0.114 0.056 13.6 31.4 Spleen 6 0.091 0.015 0.065 0.009 0.006 0.0011.4 0.2 Kidney 6 0.154 0.013 2.265 0.287 0.345 0.028 0.0 0.0 Lung 60.142 0.008 0.073 0.019 0.010 0.003 1.3 0.6 Blood 6 0.236 0.019 0.0080.005 0.013 0.007 21.0 27.9 Stomach 6 0.379 0.054 0.041 0.017 0.0160.008 2.5 1.0 Sm. Int. 6 0.870 0.042 0.137 0.031 0.119 0.029 0.7 0.3 Lg.Int. 6 0.557 0.101 0.713 0.215 0.408 0.194 0.1 0.0 Muscle 6 0.134 0.0380.013 0.007 0.002 0.001 203.9 486.6 Scapula 6 0.074 0.009 0.079 0.0260.006 0.002 1.2 0.6

TABLE 18 TF2 + Al¹⁸F(IMP485), at 1 h post peptide injection: Tissue nWeight STD WT % ID/g STD % ID/g % ID/org STD % ID/org T/NT STD T/NTTumor 7 0.291 0.134 28.089 4.545 8.025 3.357 1 0 Liver 7 1.261 0.1690.237 0.037 0.295 0.033 123 38 Spleen 7 0.081 0.013 0.254 0.108 0.0200.008 139 87 Kidney 7 0.140 0.018 3.193 0.730 0.444 0.098 9 4 Lung 70.143 0.014 0.535 0.147 0.075 0.018 57 22 Blood 7 0.205 0.029 0.2780.071 0.456 0.129 110 43 Stomach 7 0.473 0.106 0.534 1.175 0.265 0.598381 318 Sm. Int. 7 0.877 0.094 0.686 0.876 0.586 0.725 75 39 Lg. Int. 70.531 0.068 0.104 0.028 0.055 0.015 291 121 Muscle 7 0.090 0.014 0.1360.102 0.012 0.009 348 274 Scapula 6 0.189 0.029 0.500 0.445 0.095 0.092120 108

TABLE 19 TF2 + Al¹⁸F(IMP485), at 3 h post peptide injection: Tissue nWeight STD WT % ID/g STD % ID/g % ID/org STD ID/org T/NT STD T/NT Tumor7 0.320 0.249 26.518 5.971 8.127 5.181 1 0 Liver 7 1.261 0.048 0.1420.019 0.178 0.025 189 43 Spleen 7 0.079 0.012 0.138 0.031 0.011 0.002195 41 Kidney 7 0.144 0.012 2.223 0.221 0.319 0.043 12 3 Lung 7 0.1450.014 0.244 0.056 0.035 0.005 111 24 Blood 7 0.229 0.014 0.023 0.0080.037 0.012 1240 490 Stomach 7 0.430 0.069 0.025 0.017 0.010 0.005 1389850 Sm. Int. 7 0.818 0.094 0.071 0.029 0.059 0.028 438 207 Lg. Int. 70.586 0.101 0.353 0.160 0.206 0.103 86 33 Muscle 7 0.094 0.014 0.0250.006 0.002 0.001 1129 451 Scapula 7 0.140 0.030 0.058 0.018 0.008 0.002502 193

Conclusions

The IMP485 labels as well as or better than IMP467, with equivalentstability in serum. However, IMP485 is much easier to synthesize thanIMP467. Preliminary studies have shown that ¹⁸F-labeling of lyophilizedIMP485 works as well as non-lyophilized peptide (data not shown). Thepresence of alkyl or aryl groups in the linker joining the chelatingmoiety to the rest of the peptide was examined. The presence of arylgroups in the linker appears to increase the radiolabeling yieldrelative to the presence of alkyl groups in the linker.

Biodistribution of IMP485 in the presence or absence of pretargetingantibody resembles that observed with IMP467. In the absence ofpretargeting antibody, distribution of radiolabeled peptide in tumor andmost normal tissues is low and the peptide is removed from circulationby kidney excretion. In the presence of the TF2 antibody, radiolabeledIMP485 is found primarily in the tumor, with little distribution tonormal tissues. Kidney radiolabeling is substantially decreased in thepresence of the pretargeting antibody. We conclude that IMP485 and otherpeptides with aryl groups in the linker are highly suitable for PETimaging with ¹⁸F-labeling.

Example 28 Kit Formulation of IMP485 for Imaging

We report a simple, general kit formulation for labeling peptides with¹⁸F. A ligand that contains 1,4,7-triazacyclononane-1,4-diacetate (NODA)attached to a methyl phenylacetic acid (MPAA) group was used to form asingle stable complex with (AlF)²⁺. The lyophilized kit containedIMP485, a di-HSG hapten-peptide used for pretargeting. The kit wasreconstituted with an aqueous solution of ¹⁸F⁻, heated at 100-110° C.for 15 min, followed by a rapid purification by solid-phase extraction(SPE). In vitro and in vivo stability and tumor targeting of theAl¹⁸F(IMP485) were examined in nude mice bearing human colon cancerxenografts pretargeted with an anti-CEACAM5 bispecific antibody.¹⁸F-labeling of MPAA-bombesin and somatostatin peptides also wasevaluated.

The HSG peptide was labeled with ¹⁸F⁻ as a single isomer complex, inhigh yield (50-90%) and high specific activity (up to 153 GBq/μmol),within 30 min. It was stable in human serum at 37° C. for 4 h, and invivo showed low uptake (0.06%±0.02 ID/g) in bone. At 3 h, pretargetedanimals had high Al¹⁸F(IMP485) tumor uptake (26.5%±6.0 ID/g), withratios of 12±3, 189±43, 1240±490 and 502±193 for kidney, liver, bloodand bone, respectively. Bombesin and octreotide analogs were labeledwith comparable yields. In conclusion, ¹⁸F-labeled peptides can beproduced as a stable, single [Al¹⁸F] complex with good radiochemicalyields and high specific activity in a simple one-step kit.

Reagents List

Reagents were obtained from the following sources: Acetic acid (JT Baker6903-05 or 9522-02), Sodium hydroxide (Aldrich semiconductor grade99.99% 30,657-6), α,α-Trehalose (JT Baker 4226-04), Aluminum chloridehexahydrate (Aldrich 99% 237078), Ascorbic acid (Aldrich 25,556-4).

Acetate Buffer 2 mM

Acetic acid, 22.9 μL (4.0×10⁻⁴ mol) was diluted with 200 mL water andneutralized with 6 N NaOH (˜15 μL) to adjust the solution to pH 4.22.

Aluminum Solution 2 mM

Aluminum hexahydrate, 0.0225 g (9.32×10⁻⁵ mol) was dissolved in 47 mL DIwater.

α,α-Trehalose Solution

α,α-Trehalose, 4.004 g was dissolved in 40 mL DI water to make a 10%solution.

Peptide Solution, IMP485 2 mM

The peptide IMP485 (0.0020 g, 1.52 μmol) was dissolved in 762 μL of 2 mMacetate buffer. The pH was 2.48 (the peptide was lyophilized as the TFAsalt). The pH of the peptide solution was adjusted to pH 4.56 by theaddition of 4.1 μL of 1 M NaOH.

Ascorbic Acid Solution 5 mg/mL

Ascorbic acid, 0.0262 g (1.49×10⁻⁴ mol) was dissolved in 5.24 mL DIwater.

Formulation of Peptide Kit

The peptide, 20 μL (40 nmol) was mixed with 12 μL (24 nmol) of Al, 100μL of trehalose, 20 μL (0.1 mg) ascorbic acid and 900 μL of DI water ina 3 mL lyophilization vial. The final pH of the solution was about pH4.0. The vial was frozen, lyophilized and sealed under vacuum. Ten and20 nmol kits have also been made. These kits are made the same as the 40nmol kits keeping the peptide to Al³⁺ ratio of 1 peptide to 0.6 Al³⁺ butformulated in 2 mL vials with a total fill of 0.5 mL.

Purification of ¹⁸F⁻

The crude ¹⁸F⁻ was received in 2 mL of DI water in a syringe. Thesyringe was placed on a syringe pump and the liquid pushed through aWaters CM cartridge followed by a QMA cartridge. Both cartridges werewashed with 10 mL DI water. A sterile disposable three way valve betweenthe two cartridges was switched and 1 mL commercial sterile saline waspushed through the QMA cartridge in 200 μL fractions. The secondfraction usually contains ˜80% of the ¹⁸F⁻ regardless of the amount of¹⁸F⁻ applied (10-300 mCi loads were tested).

We alternatively use commercial ¹⁸F⁻ in saline, which has been purifiedon a QMA cartridge. This is a concentrated version of the commercialbone imaging agent so it is readily available and used in humans. Theactivity is supplied in 200 μL in a 0.5 mL tuberculin syringe.

Radiolabeling

The peptide was radiolabeled by adding ¹⁸F⁻ in 200 μL saline to thelyophilized peptide in a crimp sealed vial and then heating the solutionto 90-110° C. for 15 min. The peptide was purified by adding 800 mL ofDI water in a 1 mL syringe to the reaction vial, removing the liquidwith the 1 mL syringe and applying the liquid to a Waters HLB column (1cc, 30 mg). The HLB column was placed on a crimp sealed 5 mL vial andthe liquid was drawn into the vial under vacuum supplied by a remote(using a sterile disposable line) 10 mL syringe. The reaction vial waswashed with two one mL aliquots of DI water, which were also drawnthrough the column. The column was then washed with 1 mL more of DIwater. The column was then moved to a vial containing bufferedlyophilized ascorbic acid (˜pH 5.5, 15 mg). The radiolabeled product waseluted with three 200 μL portions of 1:1 EtOH/DI water. The yield wasdetermined by measuring the activity on the HLB cartridge, in thereaction vial, in the water wash and in the product vial to get thepercent yield.

Adding ethanol to the radiolabeling reaction can increase the labelingyield. A 20 nmol kit can be reconstituted with a mixture of 200 μL ¹⁸F⁻in saline and 200 μL ethanol. The solution is then heated to 110° C. inthe crimp sealed vial for 16 min. After heating, 0.8 mL of water wasadded to the reaction vial and the activity was removed with a syringeand placed in a dilution vial containing 2 mL of DI water. The reactionvial was washed with 2×1 mL DI water and each wash was added to thedilution vial. The solution in the dilution vial was applied to the HLBcolumn in 1-mL aliquots. The column and the dilution vial were thenwashed with 2×1-mL water. The radiolabeled peptide was then eluted fromthe column with 3×200 μL of 1:1 ethanol/water in fractions. The peptidecan be labeled in good yield and up to 4,100 Ci/mmol specific activityusing this method.

The yield for this kit and label as described was 80-90% when labeledwith 1.0 mCi of ¹⁸F⁻. When higher doses of ¹⁸F⁻ (˜100 mCi) were used theyield dropped. However if ethanol is added to the labeling mixture theyield goes up. If the peptides are diluted too much in saline the yieldswill drop. The labeling is also very sensitive to pH. For our peptidewith this ligand we have found that the optimal pH for the finalformulation was pH 4.0±0.2.

The purified radiolabeled peptide in 50 μL 1:1 EtOH/H₂O was mixed with150 μL of human serum and placed in the HPLC autosampler heated to 37°C. and analyzed by RP-HPLC. No detectable ¹⁸F above background at thevoid volume was observed even after 4 h.

TABLE 20 IMP 485 Labeling Activity Speci- nmol of fic of Volume VolumeActivity isolated Activ- Vial #/ Pep- Saline EtOH at start product % ityCi/ Peptide tide μL μL mCi mCi Yield mmol 1. IMP485 10 100 0 20.0 7.1062 2. IMP485 10 50 50 19.4 9.43 78 3. IMP485 10 200 0 19.07 5.05 38 4.IMP485 20 100 100 37.3 22.3 80 5. IMP485 10 100 0 45.7 16.2 42 6. IMP48520 200 200 175.6 82.7 58 4135

Synthesis and Radiolabeling of IMP486: Al—OH(IMP485)

IMP485 (21.5 mg, 0.016 mmol) was dissolved in 1 mL of 2 mM NaOAc, pH 4.4and treated with AlCl₃.6H₂O (13.2 mg, 0.055 mmol). The pH was adjustedto 4.5-5.0 and the reaction mixture was refluxed for 15 minutes. Thecrude mixture was purified by preparative RP-HPLC to yield a white solid(11.8 mg).

The pre-filled Al(NODA) complex (IMP486) was also radiolabeled inexcellent yield after formulating into lyophilized kits. The labelingyields with IMP486 (Table 21) were as good as or better than IMP485 kits(Table 20) when labeled in saline. This high efficiency of radiolabelingwith chelator preloaded with aluminum was not observed with any of theother Al(NOTA) complexes tested (data not shown). The equivalency oflabeling in saline and in 1:1 ethanol/water the labeling yields was alsonot observed with other chelating moieties (not shown).

TABLE 21 IMP486 Labeling Activity of isolated Specific Peptide VolumeVolume Activity at product Activity 20 nmol Saline EtOH start mCi mCi %Yield Ci/mmol IMP486 100 μL 0 46 28 76 2800 IMP485 100 μL 100 μL 41 2583 IMP486 100 μL 100 μL 43 22 81 IMP486 200 μL 0 42 18 73

Effect of Bulking Agents

An experiment was performed to compare yield using IMP485 kits (40 nmol)with different bulking agents. The peptide was labeled with 2 mCi of¹⁸F⁻ from the same batch of ¹⁸F in 200 microliters saline. The bulkingagents were introduced in water at a concentration of 5% by weight, witha dose of 200 microliters/vial. We tested sorbitol, trehalose, sucrose,mannitol and glycine as bulking agents. Results are shown in Table 22.

TABLE 22 Effects of Bulking Agents on Radiolabeling Yield (Refer table15) Bulking Agent Activity mCi Yield % Sorbitol 2.17 82.9 Glycine 2.1741.5 Mannitol 2.11 81.8 Sucrose 2.11 66.1 Trehalose 2.10 81.3

Sorbitol, mannitol and trehalose all gave radiolabeled product in thesame yield. The sucrose kit and the glycine kit both had significantlylower yields. Trehalose was formulated into IMP485 kits atconcentrations ranging from 2.5% to 50% by weight when reconstituted in200 μL. The same radiolabeling yield, ˜83%, was obtained for allconcentrations, indicating that the ¹⁸F-radiolabeling of IMP485 was notsensitive to the concentration of the trehalose bulking agent. IMP 485kits were formulated and stored at 2-8° C. under nitrogen for up tothree days before lyophilization to assess the impact of lyophilizationdelays on the radiolabeling. The radiolabeling experiments indicatedthat yields were all ˜80% at time zero, and with 1, 2, and 3 days ofdelay before lyophilization.

Ascorbic or gentisic acid often are added to radiopharmaceuticals duringpreparation to minimize radiolysis. When IMP485 (20 nmol) was formulatedwith 0.1, 0.5 and 1.0 mg of ascorbic acid at pH 4.1-4.2 and labeled with¹⁸F⁻ in 200 μL saline, final yields were 51, 31 and 13% isolated yields,respectively, suggesting 0.1 mg of ascorbic acid was the maximum amountthat could be included in the formulation without reducing yields.Formulations containing gentisic acid did not label well. Ascorbic acidwas also included in vials used to isolate the HLB purified product asan additional means of ensuring stability post-labeling. The IMP485 toAl³⁺ ratio appeared to be optimal at 1:0.6, but good yields wereobtained from 1:0.5 of up to a ratio of 1:1. The radiolabeling reactionwas also sensitive to peptide concentration, with good yields obtainedat concentrations of 1×10⁻⁴ M and higher.

Effect of pH on Radiolabeling

The effect of pH on radiolabeling of IMP485 is shown in Table 23. Theefficiency of labeling was pH sensitive and decreased at either higheror lower pH relative to the optimal pH of about 4.0.

TABLE 23 Effect of pH on IMP485 Radiolabeling Efficiency. pH Yield %3.27 33 3.53 61 3.84 85 3.99 88 4.21 89 4.49 80 5.07 14

Collectively, these studies led to a final formulated kit that contained0.5 mL of a sterile solution with 20 nmol IMP485, 12 nmoles Al³⁺, 0.1 mgascorbic acid, and 10 mg trehalose adjusted to 4.0±0.2, which was thenlyophilized

Biodistribution

Biodistribution studies were performed in Taconic nude mice bearingsubcutaneous LS174T tumor xenografts.

Al¹⁸F(IMP485) Alone

Mice bearing sc LS174T xenografts were injected with Al¹⁸F(IMP485) (28μCi, 5.2×10⁻¹¹ mol, 100 μL, iv). Mice were necropsied at 1 and 3 h postinjection, 6 mice per time point.

TF2+Al¹⁸F(IMP485) Pretargeting at 20:1 bsMab to Peptide Ratio

Mice bearing sc LS174T xenografts were injected with TF2 (163.2 μg,1.03×10⁻⁹ mol, iv) and allowed 16.3 h for clearance before injectingAl¹⁸F(IMP485) (28 μCi, 5.2×10⁻¹¹ mol, 100 μL, iv). Mice were necropsiedat 1 and 3 h post injection, 7 mice per time point.

TABLE 24 Biodistribution of TF2 pretargeted Al¹⁸F(IMP485) orAl¹⁸F(IMP485) alone at 1 and 3 h after peptide injection in nude micebearing LS174T human colonic cancer xenografts. Percent-injected doseper gram tissue (mean ± SD; N = 7) TF2 pregargeted Al¹⁸F(IMP485)Al¹⁸F(IMP485) alone Tissue 1 h 3 h 1 h 3 h Tumor 28.09 ± 4.55  26.52 ±5.97  0.32 ± 0.11 0.09 ± 0.02 Liver 0.24 ± 0.04 0.14 ± 0.02 0.18 ± 0.030.10 ± 0.05 Spleen 0.25 ± 0.11 0.25 ± 0.11 0.21 ± 0.18 0.07 ± 0.01Kidney 3.19 ± 0.73 2.22 ± 0.22 3.33 ± 0.56 2.27 ± 0.29 Lung 0.54 ± 0.150.24 ± 0.06 0.24 ± 0.05 0.07 ± 0.02 Blood 0.28 ± 0.07 0.02 ± 0.01 0.17 ±0.06 0.09 ± 0.01 Stomach 0.53 ± 1.18 0.03 ± 0.02 0.13 ± 0.11 0.04 ± 0.02Sm. Int. 0.69 ± 0.88 0.07 ± 0.03 0.40 ± 0.13 0.14 ± 0.03 Lg. Int. 0.10 ±0.03 0.35 ± 0.16 0.08 ± 0.02 0.71 ± 0.22 Muscle 0.14 ± 0.10 0.03 ± 0.010.11 ± 0.08 0.01 ± 0.01 Scapula  0.5 ± 0.45 0.06 ± 0.02 0.11 ± 0.02 0.03± 0.01

Synthesis of IMP492 or Al¹⁹F(IMP485)

IMP485 (16.5 mg, 0.013 mmol) was dissolved in 1 mL of 2 mM NaOAc, pH4.43, 0.5 mL ethanol and treated with AlF₃.3H₂O (2.5 mg, 0.018 mmol).The pH was adjusted to 4.5-5.0 and the reaction mixture was refluxed for15 minutes. On cooling the pH was once again raised to 4.5-5.0 and thereaction mixture refluxed for 15 minutes. The crude was purified bypreparative RP-HPLC to yield a white solid (10.3 mg).

Synthesis of IMP490

(SEQ ID NO: 52) NODA-MPAA-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Throl

The peptide was synthesized on threoninol resin with the amino acidsadded in the following order: Fmoc-Cys(Trt)-OH, Fmoc-Thr(But)-OH,Fmoc-Lys(Boc)-OH, Fmoc-D-Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Cys(Trt)-OH,Fmoc-D-Phe-OH and (tBu)₂NODA-MPAA. The peptide was then cleaved andpurified by preparative RP-HPLC. The peptide was cyclized by treatmentof the bis-thiol peptide with DMSO.

Synthesis of IMP491 or Al¹⁹F(IMP490)

The Al¹⁹F(IMP490) was prepared as described above (IMP492) to producethe desired peptide after HPLC purification.

Synthesis of IMP493

(SEQ ID NO: 53) NODA-MPAA-(PEG)3-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂

The peptide was synthesized on Sieber amide resin with the amino acidsadded in the following order: Fmoc-Met-OH, Fmoc-Leu-OH,Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Ala-OH,Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-NH-(PEG)₃-COOH and(tBu)₂NODA-MPAA. The peptide was then cleaved and purified bypreparative RP-HPLC.

The affinity of the Al¹⁹F complex of IMP493 was EC₅₀=183 nm versusEC₅₀=59 nm for ¹²⁵I-bombesin. The IMP493 kit radiolabeled with ˜100 MBqof ¹⁸F⁻ had a 70% yield. Radiolabeling IMP490 with 100 MBq of ¹⁸F⁻resulted in 80% yield, which was reduced to 65% when 2.11 GBq ¹⁸F⁻ wasused. The peptide is eluted as a single radiolabeled peak at 15.4 minusing HPLC (not shown).

Synthesis of IMP494 or Al¹⁹F(IMP493)

The Al¹⁹F(IMP493) was prepared as described above (IMP492) to producethe desired peptide after HPLC purification.

Example 29 Other Prosthetic Group Labeling Methods Using Al¹⁸F

In certain embodiments, the aluminum fluoride labeling method may beperformed using prosthetic group labeling methods for molecules that aresensitive to heat. Prosthetic group conjugation may be carried out atlower temperatures for heat-sensitive molecules.

The prosthetic group NOTA is labeled with ¹⁸F as described above andthen it is attached to the targeting molecule. In one non-limitingexample, this is performed with an aldehyde NOTA that is then attachedto an amino-oxy compound on a targeting molecule. Alternatively anamino-oxy maleimide is reacted with the aldehyde and then the maleimideis attached to a cysteine on a targeting molecule (Toyokuni et al.,2003, Bioconj Chem 14:1253).

In another alternative, the AlF-chelator complexes are attached totargeting molecules through click chemistry. The ligands are firstlabeled with Al¹⁸F as discussed above. The Al¹⁸F-chelate is thenconjugated to a targeting molecule through a click chemistry reaction.For example, an alkyne NOTA is labeled according to Marik and Stucliffe(2006, Tetrahedron Lett 47:6681) and conjugated to an azide containingtargeting agent.

Radiolabeling of Kits with ¹⁸F⁻ in Saline

The ¹⁸F⁻ (0.01 mCi or higher) is received in 200 μL of saline in a 0.5mL syringe and the solution is mixed with 200 μL of ethanol and injectedinto a lyophilized kit as described above. The solution is heated in thecrimp sealed container at 100-110° C. for 15 min. The solution isdiluted with 3 mL water and eluted through an HLB cartridge. Thereaction vial and the cartridge are washed with 2×1 mL portions of waterand then the product is eluted into a vial containing buffered ascorbicacid using 1:1 ethanol water in 0.5 mL fractions. Some of the ethanolmay be evaporated off under a stream of inert gas. The solution is thendiluted in saline and passed through a 0.2 μm sterile filter prior toinjection.

Example 30 Maleimide Conjugates of NOTA for ¹⁸F-Labeling

The Examples above describe a novel method of ¹⁸F-labeling, whichcaptures a ([¹⁸F]AlF)²⁺ complex, using a NOTA-derived ligand bound on apeptide. These labeled peptides were stable in vivo and retained theirbinding abilities (McBride et al., 2009, J. Nucl. Med. 50, 991-998;McBride et al., 2010, Bioconjug. Chem. 21, 1331-1340; Laverman et al.,2010, J. Nucl. Med. 51, 454-461; McBride et al. 2011, J. Nucl. Med. 52(Suppl. 1), 313-314P (abstract 1489)). Although this procedure allowspeptides to be radiofluorinated in one simple step within 30 min, itrequires agents to be heated to ˜100° C., which is unsuitable for mostproteins and some peptides. We and others have found that an aromaticgroup attached to one of the nitrogen atoms of the triazacyclononanering of NODA can enhance the yield for the ([¹⁸F]AlF)²⁺ complexationcompared to some alkyl and carboxyl substituents (D'Souza et al., 2011,Bioconjug. Chem. 1793-1803; McBride et al., 2010, Bioconjug. Chem. 21,1331-1340; Shetty et al. 2011, Chem. Comm. DOI: 10.1039/c1cc13151f). Inthe present Example, we explored the potential for labeling heat-labilecompounds with ([¹⁸F]AlF)²⁺, using a new ([¹⁸F]AlF)²⁺-binding ligandthat contains 1,4,7-triazacyclononane-1,4-diacetate (NODA) attached to amethyl phenylacetic acid group (MPAA). This was conjugated toN-(2-aminoethyl)maleimide (N-AEM) to form NODA-MPAEM. (Details of thesynthesis are shown in FIG. 15.) The NODA-MPAEM was labeled with([¹⁸F]AlF)²⁺ at 105° C. in 49-82% yield and conjugated at roomtemperature to an antibody Fab′ fragment in 69-80% yield (total time ˜50min) and with retention of immunoreactivity. These data indicate thatthe rapid and simple [Al¹⁸F]-labeling method can be easily adapted forpreparing heat-sensitive compounds with ¹⁸F quickly and in high yields.

Synthesis of Bis-t-Butyl-NODA-MPAA NHS Ester (tBu)₂NODA-MPAA NHS Ester

To a solution of (tBu)₂NODA-MPAA (175.7 mg, 0.347 mmol) in CH₂Cl₂ (5 mL)was added 347 μL (0.347 mmol) DCC (1 M in CH₂Cl₂), 42.5 mg (0.392mmol)N-hydroxysuccinimide (NHS), and 20 μL N,N-diisopropylethylamine(DIEA). After 3 h DCU was filtered off and solvent evaporated. The crudemixture was purified by flash chromatography on (230-400 mesh) silicagel (CH₂Cl₂:MeOH, 100:0 to 80:20) to yield (128.3 mg, 61.3%) of the NHSester. The HRMS (ESI) calculated for C₃₁H₄₆N₄O₈ (M+H)⁺ was 603.3388,observed was 603.3395.

Synthesis of NODA-MPAEM: (MPAEM=Methyl Phenyl Acetamido Ethyl Maleimide)

To a solution of (tBu)₂NODA-MPAA NHS ester (128.3 mg, 0.213 mmol) inCH₂Cl₂ (5 mL) was added a solution of 52.6 mg (0.207mmol)N-(2-aminoethyl) maleimide trifluoroacetate salt in 250 μL, DMF and20 μL, DIEA. After 3 h the solvent was evaporated and the concentratewas treated with 2 mL TFA. The crude product was diluted with water andpurified by preparative RP-HPLC to yield (49.4 mg, 45%) of the desiredproduct. HRMS (ESI) calculated for C₂₅H₃₃N₅O₇ (M+H)⁺ was 516.2453,observed was 516.2452.

¹⁸F-Labeling of NODA-MPAEM

The NODA-MPAEM ligand (20 nmol; 10 μL), dissolved in 2 mM sodium acetate(pH 4), was mixed with AlCl₃(5 μL of 2 mM solution in 2 mM acetatebuffer, 200 μL of ¹⁸F⁻ (0.73 and 1.56 GBq) in saline, and 200 μL ofacetonitrile. After heating at 105-109° C. for 15-20 mM, 800 μL ofdeionized (DI) water was added to the reaction solution, and the entirecontents removed to a vial (dilution vial) containing 1 mL of deionized(DI) water. The reaction vial was washed with 2×1 mL DI water and addedto the dilution vial. The crude product was then passed through a 1-mLHLB column, which was washed with 2×1 mL fractions of DI water. Thelabeled product was eluted from the column using 3×200 μL of 1:1EtOH/water.

Conjugation of Al¹⁸F(NODA-MPAEM) to hMN-14 Fab′

Fab′ fragments of humanized MN-14 anti-CEACAM5 IgG (labetuzumab) wereprepared by pepsin digestion, followed by TCEP(Tris(2-carboxyethyl)phosphine) reduction, and then formulated into alyophilized kit containing 1 mg (20 nmol) of the Fab′ (2.4 thiols/Fab′)in 5% trehalose and 0.025 M sodium acetate, pH 6.72. The kit wasreconstituted with 0.1 mL PBS, pH 7.01, and mixed with theAl¹⁸F(NODA-MPAEM) (600 μL 1:1 EtOH/H₂O). After incubating for 10 min atroom temperature, the product was purified on a 3-mL SEPHADEX G50-80spin column in a 0.1 M, pH 6.5 sodium acetate buffer (5 min). Theisolated yield was calculated by dividing the amount of activity in theeluent by the total activity in the eluent and the activity on thecolumn.

Immunoreactivity of the purified product was analyzed by adding anexcess of CEA and separating on SE-HPLC, comparing to a profile of theproduct alone. The product was also analyzed by RP-HPLC to assesspercent-unbound Al¹⁸F(NODA-MPAEM).

^(99m)Tc-CEA-Scan®

A CEA-SCAN® kit containing 1.2 mg of IMMU-4, a murine anti-CEACAM5 Fab′(anti-CEA, 2.4×10⁻² μmol), was labeled with 453 MBq ^(99m)TcO₄ ⁻Na⁺ in 1mL saline according to manufacturer's instructions and used withoutfurther purification.

Animal Study

Nude mice were inoculated subcutaneously with CaPan-1 human pancreaticadenocarcinoma (ATCC Accession No. HTB-79, Manassas, Va.). When tumorswere visible, the animals were injected intravenously with 100 μL of theradiolabeled Fab′. The Al¹⁸F(NODA-MPAES)-hMN-14 Fab′ was diluted insaline to 3.7 MBq/100 μL containing ˜2.8 μg of Fab′. A ^(99m)Tc-IMMU-4Fab′ aliquot (16.9 MBq) was removed and diluted with saline (0.85MBq/100 μL containing ˜2.8 μg of Fab′). The animals were necropsied at 3h post injection, tissues and tumors removed, weighed, and counted bygamma scintillation, together with standards prepared from the injectedproducts. The data are expressed as percent injected dose per gram.

Results

Synthesis and Reagent Preparation

The NODA-MPAEM was produced as shown in FIG. 15, where the(tBu)₂NODA-MPAA was coupled to 2-aminoethyl-maleimide and thendeprotected to form the desired product. The crude product was dilutedwith water and purified by preparative RP-HPLC to yield (49.4 mg, 45%)of the desired product [HRMS (ESI) calculated for C₂₅H₃₃N₅O₇ (M+H)⁺516.2453; found 516.2452].

Radiolabeling

The NODA-MPAEM (20 nmol) was mixed with 10 nmol of Al³⁺ and labeled with0.73 GBq and 1.56 GBq of ¹⁸F⁻ in saline. After SPE purification, theisolated yields of Al¹⁸F(NODA-MPAEM) were 82% and 49%, respectively,with a synthesis time of about 30 min. The Al¹⁸F(NODA-MPAES)-hMN-14 Fab′conjugate was isolated in 74% and 80% yields after spin-columnpurification for the low and high dose protein labeling, respectively.The total process was completed within 50 min. The specific activity forthe purified Al¹⁸F(NODA-MPAES)-hMN-14 Fab′ was 19.5 GBq/μmol for thehigh-dose label and 10.9 GBq/μmol for the low dose label.

SE-HPLC analysis of the labeled protein for the 0.74-GBq run showed the¹⁸F-labeled Fab′ as a single peak and all of the activity shifted whenexcess CEA was added (not shown). RP-HPLC analysis on a C4 column showedthe labeled maleimide standard eluting at 7.5 min, while the purified¹⁸F-protein eluted at 16.6 min (not shown). There was no unboundAl¹⁸F(NODA-MPAEM) in the spin-column purified product.

Serum Stability

The Al¹⁸F(NODA-MPAES)-hMN-14 Fab′ was mixed with fresh human serum andincubated at 37° C. SE-HPLC analysis over a 3-h period, with and withoutCEA showed that the product was stable and retained binding to CEA (notshown).

Biodistribution

The biodistribution of the Al¹⁸F(NODA-MPAES)-hMN-14 Fab′ and the^(99m)Tc-IMMU-4 murine Fab′ was assessed in nude mice bearing Capan-1pancreatic cancer xenografts. At 3 h post-injection, both agents showedan expected elevated uptake in the kidneys, since Fab′ is renallyfiltered from the blood (Table 25). The [Al¹⁸F]-Fab′ concentration inthe blood was significantly (P <0.0001) lower than the ^(99m)Tc-Fab′,with a correspondingly elevated uptake in the liver and spleen. Thefaster blood clearance of the [Al¹⁸F]-Fab′ likely contributed to thelower tumor uptake as compared to the ^(99m)Tc-Fab′ (2.8±0.3 vs.6.8±0.7, respectively), but it also resulted in a more favorabletumor/blood ratio for the fluorinated Fab′ (5.9±1.3 vs. 0.9±0.1,respectively). Bone uptake for both products was similar, suggesting theAl¹⁸F(NODA) was tightly held by the Fab′.

TABLE 25 Biodistribution of Al¹⁸F(NODA-MPAES)-hMN-14 Fab′ and^(99m)Tc-IMMU-4 Fab′ at 3 h after injection with 0.37 MBq (~3 μg) ofeach conjugate in nude mice bearing Capan-1 human pancreatic cancerxenografts (N = 6). Al¹⁸F(NODA-MPAES)- ^(99m)Tc CEA Scan hMN-14 Fab′IMMU-4 Fab′ Tissue % ID/g T/NT % ID/g T/NT Capan-1 2.8 ± 0.3 — 6.8 ± 0.7— (weight ± SD)  (0.22 ± 0.08 g)  (0.16 ± 0.05 g) Liver 17.5 ± 3.8   0.2± 0.04 4.6 ± 0.4 1.5 ± 0.1 Spleen 11.3 ± 1.6   0.3 ± 0.04 3.5 ± 0.5 2.0± 0.3 Kidney  216 ± 30.9 0.0 ± 0.0  183 ± 22.5 0.04 ± 0.01 Lung 4.2 ±1.6 0.8 ± 0.5 4.4 ± 0.8 1.6 ± 0.3 Blood 0.5 ± 0.1 5.9 ± 1.3 7.6 ± 0.90.9 ± 0.1 Stomach 0.6 ± 0.1 4.7 ± 1.2 2.4 ± 0.3 2.9 ± 0.4 Sm. Int. 2.2 ±0.2 1.3 ± 0.1 3.4 ± 0.4 2.0 ± 0.2 Lg. Int. 1.1 ± 0.4 2.8 ± 0.7 5.2 ± 1.01.3 ± 0.3 Muscle 0.4 ± 0.1 6.7 ± 1.3 1.1 ± 0.2 6.1 ± 1.0 Scapula 1.6 ±0.4 1.8 ± 0.4 2.0 ± 0.2 3.4 ± 0.5

Discussion

We prepared a simple NODA-MPAEM ligand for attachment to thiols ontemperature-sensitive proteins or other molecules bearing a sulfhydrylgroup. To avoid exposing the heat-labile compound to high temperatures,the NODA-MPAEM was first mixed with Al³⁺ and ¹⁸F⁻ in saline and heatedat 100-115° C. for 15 min to form the Al⁸F(NODA-MPAEM) intermediate.This intermediate was rapidly purified by SPE in 49-82% isolated yield(67.7±13.0%, n=5), depending on the amount of activity added to a fixedamount (20 nmol) of the NODA-MPAEM. The Al¹⁸F(NODA-MPAEM) was thenefficiently (69-80% isolated yield, 74.3±5.5, n=3) coupled to a reducedFab′ in 10-15 min, using a spin column gel filtration procedure toisolate the radiolabeled protein, in this case an antibody Fab′fragment. The entire two-step process was completed in ˜50 min, and thelabeled product retained its molecular integrity and immunoreactivity.Thus, the feasibility of extending the simplicity of the[Al¹⁸F]-labeling procedure to heat-sensitive compounds was established.

The [Al¹⁸F]-ligand complex has been shown to be very stable in serum invitro, and in animal testing, minimal bone uptake is seen (McBride etal., 2009, J. Nucl. Med. 50, 991-998; D'Souza et al., 2011a, J. Nucl.Med. 52 (Suppl. 1), 171P (abstract 577)). In this series of studies, ¹⁸Fassociated with the NODA-MPAEM compound conjugated to a Fab′ was stablein serum in vitro, and the conjugate retained binding to CEA. Wheninjected into nude mice, there was selective localization in the tumor,providing a ˜6:1 tumor/blood ratio. Bone uptake was similar for theAl¹⁸F(NODA-MPAES)-hMN-14 Fab′ and the ^(99m)Tc-IMMU-4 murine Fab′, againreflecting in vivo stability of the ¹⁸F or Al¹⁸F complex. However,[Al¹⁸F]-Fab′ hepatic and splenic uptake was higher as compared to the^(99m)Tc-IMMU-4. The specific NODA derivative can be modified indifferent ways to accommodate conjugation to other reactive sites onpeptides or proteins. However, use of this particular derivative showedthat the Al¹⁸F-labeling procedure can be adapted for use withheat-labile compounds.

Conclusions

NODA-MPAEM was labeled rapidly with ¹⁸F⁻ in saline and then conjugatedto the immunoglobulin Fab′ protein in high yield. The labeling methoduses only inexpensive disposable purification columns, and while notrequiring an automated device to perform the labeling and purification,it can be easily adapted to such systems. Thus, the NODA-MPAEMderivative established that this or other NODA-containing derivativescan extend the capability of facile ([¹⁸F]AlF)²⁺ fluorination toheat-labile compounds.

Example 31 Improved ¹⁸F-Labeling of NOTA-Octreotide

The aim of this study was to further improve the rapid one-step methodfor ¹⁸F-labeling of NOTA-conjugated octreotide. Octreotide wasconjugated with a NOTA ligand and was labeled with ¹⁸F in a single-step,one-pot method. Aluminum (Al³⁺) was added to ¹⁸F and the AlF²⁺ wasincorporated into NOTA-octreotide, as described in the Examples above.The labeling procedure was optimized with regard to aluminum:NOTA ratio,ionic strength and temperature. Radiochemical yield and specificactivity were determined.

Under optimized conditions, NOTA-octreotide was labeled with Al¹⁸F in asingle step with 98% yield. The radiolabeling, including purification,was performed in 45 min. Optimal labeling yield was observed withAl:NOTA ratios around 1:20. Lower ratios led to decreased labelingefficiency. Labeling efficiencies in the presence of 0%, 25%, 50%, 67%and 80% acetonitrile in Na-acetate pH 4.1 were 36%, 43%, 49%, 70% and98%, respectively, indicating that increasing concentrations of theorganic solvent considerably improved labeling efficiency. Similarresults were obtained in the presence of ethanol, DMF and THF. Labelingin the presence of DMSO failed. Labeling efficiencies in 80% MeCN at 40°C., 50° C. and 60° C. were 34%, 65%, 83%, respectively. Labelingefficiency was >98% at 80° C. and 100° C. Specific activity of the¹⁸F-labeled peptide was higher than 45,000 GBq/mmol.

Optimal ¹⁸F-labeling of NOTA-octreotide with Al¹⁸F was performed at80-100° C. in Na-acetate buffer with 80% (v/v) acetonitrile and aAl:NOTA ratio between 1:20 and 1:50. Labeling efficiency wastypically >98%. Since labeling efficiency at 60° C. was 83%, this methodmay also allow ¹⁸F-labeling of temperature-sensitive biomolecules suchas proteins and antibody fragments. These conditions allow routine¹⁸F-labeling of peptides without the need for purification prior toadministration and PET imaging.

Example 32 Functionalized Triazacyclononane Ligands for MolecularImaging

The present Example relates to synthesis and use of a new class oftriazacyclonane derived ligands and their complexes useful for molecularimaging. Exemplary structures are shown in FIG. 16 to FIG. 18. Theligands may be functionalized with a ¹⁹F moiety selected from the groupconsisting of fluorinated alkyls, fluorinated acetates, fluoroalkylphosphonates, fluoroanilines, trifluoromethyl anilines, andtrifluoromethoxy anilines in an amount effective to provide a detectable¹⁹F NMR signal. The complexation of these ligands with radioisotopic orparamagnetic cations renders them useful as diagnostic agents in nuclearmedicine and magnetic resonance imaging (MRI). Preferably, the Al¹⁸F and⁶⁸Ga complexes of these ligands are useful for PET imaging, while the¹¹¹In complexes can be used in SPECT imaging. Methods for conjugatingthese radiolabeled ligands to a targeting molecule like antibody,protein or peptide are also disclosed.

The disclosed bifunctional chelators (BFCs) can be radiolabeled with¹¹¹In, ⁶⁸Ga, ⁶⁴Cu, ¹⁷⁷Lu, Al¹⁸F, ^(99m)Tc or ⁸⁶Y or complexed with aparamagnetic metal like manganese, iron, chromium or gadolinium, andsubsequently attached to a targeting molecule (biomolecule). The labeledbiomolecules can be used to image the hematological system, lymphaticreticuloendothelial system, nervous system, endocrine and exocrinesystem, skeletomuscular system, skin, pulmonary system, gastrointestinalsystem, reproductive system, immune system, cardiovascular system,urinary system, auditory or olfactory system or to image affected cellsor tissues in various medical conditions.

Synthesis of Bifunctional Chelators2-{4-(carboxymethyl)-7-[2-(4-nitrophenyl)ethyl]-1,4,7-triazacyclononan-1-yl)aceticacid. NODA-EPN

To a solution of 4-nitrophenethyl bromide (104.5 mg, 0.45 mmol) inanhydrous CH₃CN at 0° C. was added dropwise over 20 min a solution of(tBu)₂NODA (167.9 mg, 0.47 mmol) in CH₃CN (10 mL). After 1 h, anhydrousK₂CO₃ (238.9 mg, 1.73 mmol) was added to the reaction mixture andallowed to stir at room temperature overnight. Solvent was evaporatedand the concentrate was acidified with 4 mL TFA. After 5 h, the reactionmixture was diluted with water and purified by preparative RP-HPLC toyield a pale yellow solid (60.8 mg, 32.8%). HRMS (ESI) calculated forC₁₈H₂₆N₄O₆ (M+H)⁺ 395.1925; found 395.1925.

2-{4-(carboxymethyl)-7-[2-(4-nitrophenyl)methyl]-1,4,7-triazacyclononan-1-yl)aceticacid. NODA-MPN

To a solution of 4-nitrobenzyl bromide (61.2 mg, 0.28 mmol) in anhydrousCH₃CN at 0° C. was added dropwise over 20 min a solution of (tBu)₂NODA(103.6 mg, 0.29 mmol) in CH₃CN (10 mL). After 1 h, anhydrous K₂CO₃ (57.4mg, 0.413 mmol) was added to the reaction mixture and allowed to stir atroom temperature overnight. Solvent was evaporated and the concentratewas acidified with 3 mL TFA. After 5 h, the reaction mixture was dilutedwith water and purified by preparative RP-HPLC to yield a pale yellowsolid (19.2 mg, 17.4%). HRMS (ESI) calculated for C₁₇H₂₄N₄O₆(M+H)⁺381.1769; found 381.1774.

6-(4,7-bis{[(tert-butyl)oxycarbonyl]methyl}-1,4,7-triazacyclononan-1-yl)hexanoicacid. (tBu)₂NODA-HA

To a solution of (tBu)₂NODA (208.3 mg, 0.58 mmol) in 10 mL CH₃CN wasadded 6-bromohexanoic acid (147.3 mg, 0.755 mmol) and K₂CO₃ (144.5 mg,1.05 mmol). The reaction flask was placed in a warm water-bath for 48 h.Solvent was evaporated and the concentrate was diluted with water andpurified by preparative RP-HPLC to yield a white solid (138.5 mg,50.1%). ESMS calculated for C₂₄H₄₅N₃O₆ (M+H)⁺ 472.3381; found 472.27.

4-[(4,7-bis{[(tert-butyl)oxycarbonyl]methyl}-1,4,7-triazacyclononan-1-yl)methyl]benzoicacid. (tBu)₂NODA-MBA

To a solution of α-bromo-p-toluic acid (126.2 mg, 0.59 mmol) inanhydrous CH₃CN was added dropwise over 20 min a solution of (tBu)₂NODA(208 mg, 0.58 mmol) in CH₃CN (10 mL) and allowed to stir at roomtemperature for 48 h. Solvent was evaporated and the concentrate wasdiluted with water/DMF and purified by preparative RP-HPLC to yield awhite solid (74.6 mg). HRMS (ESI) calculated for C₂₆H₄₁N₃O₆(M+H)⁺492.3068; found 492.3071.

4-[(4,7-bis{[(tert-butyl)oxycarbonyl]methyl}-1,4,7-triazacyclononan-1-yl)ethyl]benzoicacid. (tBu)₂NODA-EBA

To a solution of 4-(2-bromoethyl)benzoic acid (310.9 mg, 1.36 mmol) inanhydrous CH₃CN was added dropwise over 20 min a solution of (tBu)₂NODA(432.3 mg, 1.21 mmol) in CH₃CN (10 mL) and K₂CO₃ (122.4 mg, 0.89 mmol).The reaction was stirred at room temperature for 72 h. Solvent wasevaporated and the concentrate was diluted with water/DMF and purifiedby preparative RP-HPLC to yield a white solid (35.1 mg). HRMS (ESI)calculated for C₂₇H₄₃N₃O₆ (M+H)⁺ 506.3225; found 506.3234.

2-[7-but-3-ynyl-4-(carboxymethyl]-1,4,7-triazacyclononan-1-yl)aceticacid. NODA-Butyne

To a solution of (tBu)₂NODA (165.8 mg, 0.46 mmol) in 5 mL CH₃CN wasadded 4-bromo-1-butyne (44 μL, 62.3 mg, 0.47 mmol) and reaction mixturewas stirred at room temperature for 72 h. Solvent was evaporated and theconcentrate was purified by preparative RP-HPLC to yield an oil. HRMS(ESI) calculated for C₂₂H₃₉N₃O₄ (M+H)⁺410.3013; found 410.3013. Thepurified product was acidified with 2 mL TFA and after 5 h diluted withwater, frozen and lyophilized. HRMS (ESI) calculated for C₁₄H₂₃N₃O₆(M+H)⁺298.1761; found 298.1757.

tert-butyl-2-(7-(4-aminobutyl)-4-{[(tert-butyl)oxycarbonyl]methyl}-1,4,7-triazacyclononan-1-yl)aceticacid. (tBu)₂NODA-BA

To a solution of (tBu)₂NODA (165.2 mg, 0.46 mmol) in 5 mL CH₃CN wasadded 4-(Boc-amino)butyl bromide (124.7 mg, 0.49 mmol), a pinch of K₂CO₃and reaction mixture was stirred at room temperature for 72 h. Solventwas evaporated and the concentrate was treated with 1 mL CH₂Cl₂ and 0.5mL TFA. After 5 min the solvents were evaporated and the crude oil wasdiluted with water/DMF and purified by preparative RP-HPLC to yield awhite solid′(137.2 mg, 69.3%). HRMS (ESI) calculated for C₂₂H₄₄N₄O₄(M+H)⁺ 429.3435; found 429.3443.

NODA-BAEM: (BAEM=Butyl Amido Ethyl Maleimide)

To a solution of (tBu)₂NODA-BM (29.3 mg, 0.068 mmol) in CH₂Cl₂ (3 mL)was added a β-maleimido propionic acid NHS ester (16.7 mg, 0.063 mmol),20 μL DIEA and stirred at room temperature overnight. Solvent wasevaporated and the concentrate was acidified with 1 mL TFA. After 3 h,the reaction mixture was diluted with water and purified by preparativeRP-HPLC to yield a white solid. HRMS (ESI) calculated for C₂₁H₃₃N₅O₇(M+H)⁺ 468.2453; found 468.2441.

2-{4-[(4,7-bis-tert-butoxycarbonylmethyl)-[1,4,7]-triazacyclononan-1-yl)methyl]phenyl}aceticacid. (tBu)₂NODA-MPAA

To a solution of 4-(bromomethyl)phenylacetic acid (593 mg, 2.59 mmol) inanhydrous CH₃CN (50 mL) at 0° C. were added dropwise over 1 h a solutionof (tBu)₂NODA (1008 mg, 2.82 mmol) in CH₃CN (50 mL). After 4 h,anhydrous K₂CO₃ (100.8 mg, 0.729 mmol) was added to the reaction mixtureand allowed to stir at room temperature overnight. Solvent wasevaporated and the crude was purified by preparative RP-HPLC (Method 5)to yield a white solid (713 mg, 54.5%). ¹H NMR (500 MHz, CDCl₃, 25° C.,TMS) δ 1.45 (s, 18H), 2.64-3.13 (m, 16H), 3.67 (s, 2H), 4.38 (s, 2H),7.31 (d, 2H), 7.46 (d, 2H); ¹³C (125.7 MHz, CDCl₃) δ 28.1, 41.0, 48.4,50.9, 51.5, 57.0, 59.6, 82.3, 129.0, 130.4, 130.9, 136.8, 170.1, 173.3.HRMS (ESI) calculated for C₂₇H₄₃N₃O₆ (M+H)⁺ 506.3225; found 506.3210.

2-(4-(carboxymethyl)-7-{[4-(carboxymethyl)phenyl]methyl}-1,4,7-triazacyclononan-1-yl)aceticacid. NODA-MPAA

To a solution of 4-(bromomethyl)phenylacetic acid (15.7 mg, 0.068 mmol)in anhydrous CH₃CN at 0° C. was added dropwise over 20 min a solution of(tBu)₂NODA (26 mg, 0.073 mmol) in CH₃CN (5 mL). After 2 h, anhydrousK₂CO₃ (5 mg) was added to the reaction mixture and allowed to stir atroom temperature overnight. Solvent was evaporated and the concentratewas acidified with 2 mL TFA. After 3 h, the reaction mixture was dilutedwith water and purified by preparative RP-HPLC to yield a white solid(11.8 mg, 43.7%). ¹H NMR (500 MHz, DMSO-d₆, 25° C.) δ 2.65-3.13 (m,12H), 3.32 (d, 2H), 3.47 (d, 2H), 3.61 (s, 2H), 4.32 (s, 2H), 7.33 (d,2H), 7.46 (d, 2H); ¹³C (125.7 MHz, DMSO-d₆) 40.8, 47.2, 49.6, 50.7,55.2, 58.1, 130.4, 130.5, 130.9, 136.6, 158.4, 158.7, 172.8, 172.9. HRMS(ESI) calculated for C₁₉H₂₇N₃O₆ (M+H)⁺ 394.1973; found 394.1979.

(tBu)₂NODA-MPAA NHS ester.

To a solution of (tBu)₂NODA-MPAA (175.7 mg, 0.347 mmol) in CH₂Cl₂ (5 mL)was added (1 M in CH₂Cl₂) DCC (347 μL, 0.347 mmol), N-hydroxysuccinimide(NHS) (42.5 mg, 0.392 mmol), and 20 μL N,N-diisopropylethylamine (DIEA).After 3 h, dicyclohexylurea (DCU) was filtered off and solventevaporated. The crude product was purified by flash chromatography on(230-400 mesh) silica gel (CH₂Cl₂:MeOH (100:0 to 80:20) to yield the NHSester (128.3 mg, 61.3%). HRMS (ESI) calculated for C₃₁H₄₆N₄O₈ (M+H)⁺603.3388; found 603.3395.

NODA-MPAEM: (MPAEM=Methyl Phenyl Acetamido Ethyl Maleimide)

To a solution of (tBu)₂NODA-MPAA NHS ester (128.3 mg, 0.213 mmol) inCH₂Cl₂ (5 mL) was added a solution of N-(2-aminoethyl) maleimidetrifluoroacetate salt (52.6 mg, 0.207 mmol) in 250 μL DMF and 20 μLDIEA. After 3 h, the solvent was evaporated and the concentrate treatedwith 2 mL TFA. The crude product was diluted with water and purified bypreparative RP-HPLC to yield a white solid (49.4 mg, 45%). HRMS (ESI)calculated for C₂₅H₃₃N₅O₇ (M+H)⁺516.2453; found 516.2452.

tert-butyl-2-(7-(4-aminopropyl)-4-{[(tert-butyl)oxycarbonyl]methyl}-1,4,7-triazacyclononan-1-yl)aceticacid. (tBu)₂NODA-PA

To a solution of (tBu)₂NODA (391.3 mg, 1.09 mmol) in 5 mL CH₃CN wasadded Benzyl-3-bromo propyl carbamate (160 μL) and reaction mixture wasstirred at room temperature for 28 h. Solvent was evaporated and theconcentrate was dissolved in 40 mL 2-propanol, mixed with 128.7 mg of10% Pd—C and placed under 43 psi H₂ overnight. The product was thenfiltered and the filtrate concentrated. The crude product was dilutedwith water/DMF and purified by preparative RP-HPLC to yield a whitesolid (353 mg). HRMS (ESI) calculated for C₂₁H₄₂N₄O₄ (M+H)⁺ 415.3291;found 415.3279.

NODA-PAEM: (PAEM=Propyl Amido Ethyl Maleimide)

To a solution of (tBu)₂NODA-PM (109.2 mg, 0.263 mmol) in CH₂Cl₂ (3 mL)was added a β-maleimido propionic acid NHS ester (63.6 mg, 0.239 mmol),20 μL DIEA and stirred at room temperature overnight. Solvent wasevaporated and the concentrate was acidified with 1 mL TFA. After 3 h,the reaction mixture was diluted with water and purified by preparativeRP-HPLC to yield a white solid (79 mg). HRMS (ESI) calculated forC₂₀H₃₁N₅O₇ (M+H)⁺ 454.2319; found 454.2296.

¹⁸F-Labeling of Functionalized Triazacyclononane Ligands

The functionalized triazacyclononane ligand (20 nmol; 10 μL), dissolvedin 2 mM sodium acetate (pH 4), was mixed with AlCl₃ (5 μL of 2 mMsolution in 2 mM acetate buffer, 25-200 μL of ¹⁸F⁻ in saline, and 25-200μL of ethanol. After heating at 90-105° C. for 15-20 min, 800 μL ofdeionized (DI) water was added to the reaction solution, and the entirecontents removed to a vial (dilution vial) containing 1 mL of deionized(DI) water. The reaction vial was washed with 2×1 mL DI water and addedto the dilution vial. The crude product was then passed through a 1-mLHLB column, which was washed with 2×1 mL fractions of DI water. Thelabeled product was eluted from the column using 3×200 μL of 1:1EtOH/water. Radiochromatograms of the ¹⁸F-labeling of functionalizedTACN ligands are shown in FIG. 19.

¹⁸F-labeling of NODA-MPAEM

To 10 μL (20 nmol) 2 mM NODA-MPAEM solution was added 5 μL 2 mM AlCl₃,200 μL ¹⁸F⁻ solution [15.94 mCi, Na¹⁸F, PETNET] followed by 200 μL CH₃CNand heated to 110° C. for 15 minutes. The crude reaction mixture waspurified by transferring the resultant solution into a Oasis® HLB 1 cc(30 mg) cartridge (P#186001879, L#099A30222A) and eluting with DI H₂O toremove unbound ¹⁸F⁻ followed by 1:1 EtOH/H₂0 to elute the ¹⁸F-labeledpeptide. The crude reaction solution was pulled through the HLBcartridge into a 10 mL vial and the cartridge washed with 6×1 mLfractions of DI H₂O (4.34 mCi). The HLB cartridge was then placed on anew 3 mL vial and eluted with 4×150 μL 1:1 EtOH/H₂O to collect thelabeled peptide (7.53 mCi). The reaction vessel retained 165.1 μCi,while the cartridge retained 270 μCi of activity. 7.53 mCi

61.2% of Al[¹⁸F]NODA-MPAEM.

¹⁸F-Labeling of NODA-MPAEM:

To 10 μL (20 nmol) 2 mM NODA-MPAEM solution was added 5 μL 2 mM AlCl₃,200 μL ¹⁸F⁻ solution [15.94 mCi, Na¹⁸F, PETNET] followed by 200 μL CH₃CNand heated to 110° C. for 15 minutes. The crude reaction mixture waspurified by transferring the resultant solution into a Oasis® HLB 1 cc(30 mg) cartridge (P#186001879, L#099A30222A) and eluting with DI H₂O toremove unbound ¹⁸F⁻ followed by 1:1 EtOH/H₂O to elute the ¹⁸F-labeledpeptide. The crude reaction solution was pulled through the HLBcartridge into a 10 mL vial and the cartridge washed with 6×1 mLfractions of DI H₂O (4.34 mCi). The HLB cartridge was then placed on anew 3 mL vial and eluted with 4×150 μL 1:1 EtOH/H₂O to collect thelabeled peptide (7.53 mCi). The reaction vessel retained 165.1 μCi,while the cartridge retained 270 μCi of activity. 7.53 mCi

61.2% of Al[¹⁸F]NODA-MPAEM.

TABLE 26 ¹⁸F-labeling of 20 nmol NODA-MPAEM + 10 nmol Al³⁺ Activity^(a)Na¹⁸F Aqueous CH₃CN Isolated activity^(b) RCY^(c) (mCi) (μL) (μL) (mCi)(%) 2.02 80 — 1.09 64.1 1.86 40 40 1.43 91.0 1.96 50 50 1.371 90.8 3.26200 200 2.05 76.0 15.08 200 200 8.24 70.3 15.94 200 200 7.53 61.2 ^(a)10μL NODA-MPAEM, 5 μL Al³⁺, 105-110° C., 15 min. ^(b)Isolated activity in(1:1) EtOH/H₂O after HLB column purification (SPE). ^(c)decay correctedRCY - based on synthesis time of 27-42 minutes.

Conjugation of hMN14-Fab′-SH with Al¹⁸F(NODA-MPAEM):

To the vial containing hMN14-Fab′-SH (1 mg, ˜20 nmoles) L #112310 wasadded 200 μL PBS, pH 7.38 and 600 μL of the HLB purifiedAl¹⁸F(NODA-MPAEM) (EtOH:H₂O::1:1). The crude reaction mixture was passedthrough a sephadex (G-50/80, 0.1 M NaOAc, pH 6.5) 3 mL spin column. Theactivity in the eluate was 4.27 mCi, while 1.676 mCi was retained on thespin column and 0.178 mCi in the empty reaction vial. 4.27 mCi

68.6% of [Al¹⁸F]-hMN14-Fab.

To 800 μL of PBS was added 1 μL of eluate

injected 40 μL (SEC-HPLC) at 2.08 p.m. Major product at 10.312 min.

Serum Stability:

In an autosampler vial 200 μL of fresh human serum+50 μL of eluate

149.6 μCi at 2.45 p.m. Incubated at 37° C.

[Al¹⁸F]-hMN14-Fab in serum.

To 4 μL of [Al¹⁸F]-hMN14-Fab in serum added 280 μL of buffer B (PBS)

injected 40 μL (SEC-HPLC) at 3.51 p.m. Major product at 10.331 min.

Immunoreactivity of ¹⁸F-hMN14-Fab:

To 100 μg carcinoembryonic antigen (CEA) [L #2371505, Scripp's Labs] wasadded 200 μL 1% HSA in PBS, pH 7.38+100 μL PBS, pH 7.38

CEA in PBS. Added 4 μL of [Al¹⁸F]-hMN14-Fab in serum at 37° C. to 150 μLCEA in PBS

injected 40 μL (SEC-HPLC) at 4.33 p.m. Major product at 7.208 min.

Radiochromatograms of spin column purified [Al¹⁸F]-hMN14-Fab, stabilityof [Al¹⁸F]-hMN14-Fab in human serum and its immunoreactivity with CEAare shown in FIG. 20.

Exemplary synthetic schemes for the bifunctional chelators are shownbelow.

Exemplary structures of ¹⁸F-labeled probes are shown below.

Conclusions

We have found that a novel class of triazacyclononane (TACN) derivedBFCs, possessing a functionality that provides for an easy linkage ontobiomolecules via solid phase or in solution, form stable complexes witha variety of metals. These BFCs also form remarkably stable Al¹⁸Fchelates. Most ¹⁸F-labeling methods are tedious to perform, require theefforts of a specialized chemist, involve multiple purifications of theintermediates, anhydrous conditions, and generally end up with low RCYs.An advantage of this new class of BFCs is that they can beradiofluorinated rapidly in one step with high specific activity in anaqueous medium.

Example 33 Further Optimization of Kit Formulation

The effect of varying buffer composition on labeling efficiency wasdetermined. Kits were formulated with 20 nmol IMP485 and 10 nmolAlCl₃.6H₂O in 5% α,α-trehalose. The buffers and ascorbic acid werevaried in the different formulations. The peptide and trehalose weredissolved in DI water and the AlCl₃.6H₂O was dissolved in the buffertested.

MES Buffer

4-morpholineethanesulfonic acid (MES, Sigma M8250), 0.3901 g (0.002 mol)was dissolved in 250 mL of DI H₂O and adjusted to pH 4.06 with aceticacid (8 mM buffer).

KHP Buffer

Potassium biphthalate (KHP, Baker 2958-1), 0.4087 g (0.002 mol) wasdissolved in 250 mL DI H₂O pH 4.11 (8 mM buffer).

HEPES Buffer

N-2 hydroxyethylpiperazine-N′-2-ethane-sulfonic acid (HEPES, Calbiochem391338) 0.4785 g (0.002 mol) dissolved in 250 mL DI H₂O and adjusted topH 4.13 with AcOH (8 mM buffer).

HOAc Buffer

Acetic acid (HOAc, Baker 9522-02), 0.0305 g (0.0005 mol) was dissolvedin 250 mL DI H₂O and adjusted to pH 4.03 with NaOH (2 mM buffer).

The AlCl₃.6H₂O (Aldrich 23078) was dissolved in the buffers to obtain a2 mM solution of Al³⁺ in 2 mM buffer. IMP 485 0.0011 g (MW 1311.67,8.39×10⁻⁷ mol) was dissolved in 419 μL DI H₂O. Ascorbic acid, 0.1007 g(Aldrich 25,556-4, 5.72×10⁻⁴ mol) was dissolved in 20 mL DI H₂O.

A variety of kits (summarized in Table 27) were prepared and adjusted tothe proper pH by the addition of NaOH or HOAc as needed. The solutionwas then dispensed in 1 mL aliquots into 4, 3 mL lyophilization vials,frozen on dry ice and lyophilized. The initial shelf temperature for thelyophilization was −10° C. The samples were placed under vacuum and theshelf temperature was increased to 0° C. The samples were lyophilizedfor 15 hr and the shelf temperature was increased to 20° C. for 1 hbefore the vials were sealed under vacuum and removed from thelyophilizer. The kits were prepared with different buffers, at differentpH values, with or without ascorbic acid and with or without acetate.After lyophilization, the kits were dissolved in 400 μL of saline andthe pH was measured with a calibrated pH meter with a micro pH probe.

Radiolabeling

The kits were all labeled with ¹⁸F⁻ in saline (200 μL, PETNET) withethanol (200 μL) and heated to ˜105° C. for 15 min. The labeled peptideswere diluted with 0.6 mL DI H₂O and then added to a dilution vialcontaining 2 mL DI H₂O. The reaction vial was washed with 2×1 mLportions of DI H₂O, which were added to the dilution vial. The dilutedsolution was filtered through a 1 mL (30 mg) HLB cartridge (1 mL at atime) and washed with 2 mL DI H₂O. The cartridge was moved to an emptyvial and eluted with 3×200 μL 1:1 EtOH/DI H₂O. The Al[¹⁸F]IMP485 was inthe 1:1 EtOH/DI H₂O fractions. The isolated yield was determined bycounting the activity in the reaction vial, the dilution vial, the HLBcartridge, the DI H₂O column wash and the 1:1 EtOH/DI H₂O wash adding upthe total and then dividing the amount in the 1:1 EtOH/DI H₂O fractionby the total and multiplying by 100.

Results

The results of the studies are shown in Table 27. All the labeling inthe presence of 0.1 mg of ascorbic acid went well. The ascorbic acidappears to serve as a significant non-volatile buffer that keeps the pHthe same before and after lyophilization (kits 1-4). When ascorbic acidis not used (kits 5-8) the pH can change significantly along with theradiolabeling yield. The KHP buffer, kit 8, was the best kit in thesecond batch. Higher levels of ascorbate might also stabilize the Al¹⁸Fcomplex in solution and act as a transfer ligand for Al¹⁸F. The KHPbuffer might also act as a transfer ligand for Al¹⁸F so the amount ofKHP was increased from 5×10⁻⁷ mol/kit for kit 8 to 6×10⁻⁶ mol/kit forkit 11. The increase in KHP stabilized the pH better than kit 8 and gavea much better labeling yield. The kits with KHP+ascorbate (kit 12) andKHP+MES (kit 13) had slightly higher labeling yields. It may be that thehigher levels of KHP and ascorbate act both as buffers and as transferligands to increase the labeling yields with those excipients. Citricacid is not a good buffer for [Al¹⁸F]-labeling (kit 14), it gives lowlabeling yields even when only 50 μL of 2 mM citrate was used in thepresence 0.1 mg of ascorbate. Increasing amounts of KHP, 0.1 M and above(kits 16-18) lead to lower labeling yields with more activity found inthe aqueous wash from the HLB column.

TABLE 27 Results of labeling and pH studies pH before pH after lyophili-lyophili- Isolated Kit/lot Buffer zation zation % yield 1. HEPES +ascorbic 4.07 3.83 82.6 2. MES + ascorbic 4.08 3.99 83.5 3. NaOAc +ascorbic 4.10 4.10 83.5 4. KHP + ascorbic 4.12 4.10 86.1 5. HEPES Noascorbic + HOAc 4.10 4.24 45.4 6. MES No ascorbic + HOAc 4.07 4.25 66.47. HOAc No ascorbic + HOAc 4.11 4.50 35.2 8. KHP No ascorbic + HOAc 4.014.09 71 9. HEPES No ascorbic or NaOAc 4.07 4.44 69 10. MES No ascorbicor NaOAc 4.06 4.62 75 11. BM 20-57 KHP alone 0.015M 4.07 4.05 83.9 12.BM 20-57 KHP 0.015M + 4.03 3.96 85.8 ascorbic 13. BM 20-57 KHP 0.015M +4.10 4.09 87.0 MES 0.015M 14. Citric acid 4.03 3.93 31.2 15. Ascorbicacid 4.13 4.04 80.0 16. KHP 0.1M 4.09 3.82 80.7 17. KHP 0.2M 4.05 3.7978.1 18. KHP 0.4M 4.02 3.79 69.0

It appears from these results that potassium biphthalate is an optimalbuffer for labeling. The peptide labeling kits were thereforereformulated to utilize KHP in the labeling buffer. The reformulatedkits gave very high isolated labeling yields of about 97% when 100 nmolof peptide was labeled in 1:1 ethanol/saline. The labeling andpurification time was also simplified and reduced to 20 min. In additionto using the new buffering agent, potassium biphthalate (KHP), we alsoadded more moles of buffer, which may help stabilize the pH duringlabeling. The peptide is purified through an Alumina N cartridge byadding more saline to the reaction after heating and pushing crudeproduct through the cartridge. The unbound ¹⁸F⁻ and Al¹⁸F stick to thealumina and the labeled peptide is eluted very efficiently from thecartridge with saline. The formulation shown below is for a 20 nmolpeptide kit but the same formulation is used for a 100 nmol peptide kitby adding more peptide and more Al³⁺ (60 nmol Al³⁺ for the 100 nmolpeptide kit).

2 mM Al³⁺ in 2 mM KHP

Aluminum chloride hexahydrate, 0.0196 g (8.12×10⁻⁵ mol, Aldrich 23078,MW 241.43) was dissolved in 40.6 mL of 2 mM potassium biphthalate (KHP,JT Baker 2958-1, MW 204.23). This can be stored at room temperature formonths.

Ascorbic Acid

Ascorbic acid, 0.100 g was dissolved in 20 mL DI H₂O. This is made freshon the day of use.

5% Trehalose α,α-Trehalose dihydrate, 2.001 g (JT Baker, 4226-04, MW378.33) was dissolved in 20 mL DI H₂O. This can be stored at roomtemperature for weeks.

KHP Kit Buffer

KHP, 0.2253 g was dissolved in 18 mL DI H₂O (0.06 M). This solution canbe kept for months at room temperature.

IMP485 Solution

IMP485, 0.0049 g (3.74×10⁻⁶ mol, MW 1311.67) was dissolved in 1.494 mLDI H₂O (2.5×10⁻³ M). This solution can be stored for months at −20° C.

1M KOH

Potassium hydroxide (99.99% semiconductor grade, MW 56.11, Aldrich306568) was dissolved in DI H₂O to make a 1 M solution.

Kit Formulation (20 nmol Kit, 40 Kits)

The peptide, IMP485 (320 μL, 8×10⁻⁷ mol) was placed in a 50 mL sterilepolypropylene centrifuge tube (metal free) and mixed with 240 μL of the2 mM Al³⁺ solution (4.8×10⁻⁷ mol) 800 μL of the ascorbic acid solution,1600 μL of the 0.06 M KHP solution, 8 mL of the 5% trehalose solutionand the mixture was diluted to 40 mL with DI H₂O. The solution wasadjusted to pH 3.99-4.03 with a few microliters of 1 M KOH. The peptidesolution was dispensed 1 mL/vial with a 1 mL pipette into 3 mL glasslyophilization vials (unwashed).

Lyophilization

The vials were frozen on dry ice, fitted with lyophilization stoppersand placed on a −20° C. shelf in the lyophilizer. The vacuum pump wasturned on and the shelf temperature was raised to 0° C. after the vacuumwas below 100 mtorr. The next morning the shelf temperature was raisedto 20° C. for 4 hr before the samples were closed under vacuum and crimpsealed.

Radiolabeling

The ¹⁸F⁻ in saline was received from PETNET in 200 μL saline in a 0.5 mLtuberculin syringe. Ethanol, 200 μL, was pulled into the ¹⁸F⁻ solutionand then the mixture was injected into a lyophilized kit containing thepeptide. The solution was then heated in a 105° C. heating block for 15mM. Sterile saline, 0.6 mL was then added to the reaction vial and thesolution was removed from the vial and pushed through an alumina Ncartridge (SEP-PAK light, WAT023561, previously washed with 5 mL sterilesaline) into a collection vial. The reaction vial was washed with 2×1 mLsaline and the washes were pushed through the alumina column. The totallabeling and purification time was about 20 min.

Example 34 Labeling at Reduced Temperature

The effect of varying the chelator structure on efficiency of labelingat reduced temperature was examined. A comparison of low temperaturelabeling of IMP466 (NOTA-Octreotide) with IMP485 showed that the simpleNOTA ligand labels much better at low temperature than the NODA-MPAAligand.

In one embodiment, a temperature sensitive molecule, such as a protein,may be conjugated to multiple copies of a simple NOTA ligand. Theprotein can then be purified and formulated for Al¹⁸F-labeling (e.g.,lyophilized). The protein kit was reconstituted with ¹⁸F⁻ in saline,heated for the appropriate length of time and purified by gel filtrationor an alumina column. Tables 27 and 28 show the temperature effects oflabeling IMP466 vs. IMP485.

TABLE 28 Temperature-dependent labeling for Al¹⁸F(IMP466) % Yield %Yield % Yield % Yield % Yield Temp ° C. 25 μM 50 μM 100 μM 250 μM 500 μM50 3.3 8.6 14.5 20.5 37.1 70 21.3 47.4 58.0 82.8 93.6 90 29.0 50.7 70.383.0 93.5 100 34.3 55.8 77.4 84.0 94.5 110 34.9 60.3 78.1 87.9 90.5

TABLE 29 Temperature-dependent labeling for Al¹⁸F(IMP485) % Yield %Yield % Yield % Yield % Yield Temp ° C. 25 μM 50 μM 100 μM 250 μM 500 μM50 1.31 3.29 3.18 6.10 12.99 70 7.01 12.8 22.90 36.8 39.8 90 22.2 38.382.3 85.4 85.3 100 48.6 76.1 91.8 94.6 96.6 110 61.6 74.4 96.4 94.0 96.8

The data show that by switching to a different chelating moiety, theefficiency of low temperature labeling with Al¹⁸F may be tripled at 50°C. Further modification of the chelating moiety may provide additionalimprovement of low temperature labeling. However, the 37% efficiencyobserved with IMP466 is sufficient to enable ¹⁸F PET imaging withtemperature sensitive molecules if a sufficient number of chelatingmoieties are attached to the molecule.

We have also examined the effect of peptide concentration on lowtemperature labeling of IMP485. Kits were made with 10, 20, 40, 100 and200 nmol of peptide and 0.6 equivalents of Al³⁺ respectively. The restof the formulation was the same for all of the kits. The kits werelabeled with 400 μL saline/EtOH and heated at 50-110° C. for 15 min andthen purified through the Alumina N cartridge. The labeling results arereported as isolated yields in Table 30. At any temperature, increasingthe concentration of peptide increased the efficiency of labeling. Theresults indicate that if the reaction volume can be decreased with theuse of a microfluidics device then we can greatly reduce the amount ofpeptide and ¹⁸F⁻ needed to prepare a single dose of labeled peptide forPET imaging.

TABLE 30 Effect of peptide concentration on efficiency of labeling as afunction of temperature. % Yield % Yield % Yield % Yield % Yield Temp °C. 25 μM 50 μM 100 μM 250 μM 500 μM 50 1.31 3.29 3.18 6.10 12.99 70 7.0112.8 22.90 36.8 39.8 90 22.2 38.3 82.3 85.4 85.3 100 48.6 76.1 91.8 94.696.6 110 61.6 74.4 96.4 94.0 96.8

Example 35 Automated Synthesis of ¹⁸F-Labeled Molecules

This Example compared the automated synthesis of ¹⁸F-FBEM published byKiesewetter et al., (2011, Appl Radiat Isot 69:410-4) to that ofAl¹⁸F(NODA-MPAEM). The automated synthesis of ¹⁸F-FBEM was accomplishedusing a sophisticated synthesis module (see below), with a RCY of 17% in95 min. Our synthesis module (FIG. 21) would include a heating deviceand a HLB cartridge or HPLC column. With NODA-MPAEM we were able to get67-79% RCY (decay corrected) in 40 min in one single step.

In both ¹⁸F-FBEM and ¹⁸F-FDG-MHO, the ¹⁸F is introduced first followedby a maleimide (Scheme 20 and 21). While NODA-MPAEM—a maleimidecontaining BFC—is ¹⁸-labeled in one final step (Scheme 19).

Prosthetic Synthesis Synthesis RCY Specific group module time (%)activity ¹⁸F-SFB TRACERlab ™ 98 min 44.3 ± 2.5 250-350 8-12 GBq MX_(FDG)GBq/μmol (216-324 (6.8-9.5 mCi) Ci/μmol) Prosthetic Synthesis SynthesisRCY* Specific group module time (%) activity ¹⁸F-FBEM Eckert & 95 min17.3 ± 7.1 181-351 Ziegler GBq/μmol (4.9-9.5 Ci/μmol) With 8.2 GBq (222mCi) ¹⁸F⁻ provide 1.87 GBq (50.6 mCi) of ¹⁸F-FBEM in 96 min (22.8%uncorrected; 41.7% corrected for decay). *not decay corrected.

Example 34 Room Temperature Labeling of Targeting Molecules UsingBifunctional Chelator (BFC) Moieties

The objective of this Example was to perform ¹⁸F-labeling of temperaturesensitive molecules at reduced temperatures, such as room temperature,with high radiochemical yield and high specific activity of the labeledmolecule. Preferably, the labeling reaction is accomplished in 10 to 15minutes in aqueous medium, with a total synthesis time of 30 minutes orless. More preferably, the labeling technique involves the initialreaction of a metal-¹⁸F or metal-¹⁹F with a bifunctional chelating (BFC)moiety at elevated temperature (e.g., 90 to 105° C.), followed bysite-specific attachment of the BFC to the targeting molecule at areduced temperature (e.g., room temperature). In certain embodiments,the BFC may be derived from the structure of NODA-propyl amine (FIG.22).

IMP508 (FIG. 23A) and IMP517 (FIG. 23B) were synthesized as disclosedbelow. The NODA chelating moiety formed according to schemes 22 and 23was attached to a bis-HSG peptide (IMP508), formulated into 20 nmolpeptide kits and labeled with ¹⁸F.

The methyl ester was synthesized as follows. The NO₂AtBu, 1.0033 g(2.807×10⁻³ mol) was mixed with 0.4638 g (2.810×10⁻³ mol) of the methyl6-formylnicotinate and dissolved in 10 mL THF. Triacetoxyborohydride,0.6248 g (2.948×10⁻³ mol) was added and the reaction was stirred at roomtemperature for two days and an additional 0.3044 g of the borohydridewas added. The reaction was quenched with H₂O after stirring 6.5 hr moreat room temp. The product was extracted with dichloromethane, dried overNa₂SO₄, filtered and concentrated under reduced pressure to obtain thecrude brown product. The product was purified by flash chromatographyeluting with hexanes, 25% EtOAc/hexanes, 50% EtOAc/hexanes, 75%EtOAc/hexanes, 100% EtOAc, dichloromethane, 5% MeOH/94%dichloromethane/1% triethylamine and 10% MeOH/89% dichloromethane/1%triethylamine. The product, was isolated as a brown tar 0.455 g and wasin the MeOH/dichloromethane/triethylamine fractions.

To synthesize the acid, the methyl ester (0.411 g, 8.12×10⁻⁴ mol) wasdissolved in 5 mL dioxane and stirred with 0.8 mL of 1 M NaOH. Thereaction was stirred for 18 hr at room temperature and another 1.3 mL ofNaOH was added in portions as the reaction stirred at room temperaturefor another 8 hr. The reaction was quenched with 1 M citric acid andadjusted to pH 4.91 with 1 M NaOH. The product was extracted withdichloromethane. Some saturated NaCl solution was added to the aqueouslayer and the solution was again extracted with dichloromethane. Theorganic layers were combined, dried over Na₂SO₄, and concentrated toobtain 0.3421 g of the product (85% yield).

IMP517 was produced as disclosed in Scheme 24. The methyl ester triazoleprecursor was hydrolyzed and conjugated to the bis-HSG peptide to obtainIMP517 (FIG. 23B).

IMP517 was test labeled with different concentrations of peptide in 400μL of saline. IMP485 was also labeled in 400 μL of saline forcomparison.

Peptide/nmol labeled in 400 μL Isolated % saline 110° C., 15 min YieldIMP517 2.5 nmol 5.54 IMP517 5 nmol 31.1 IMP517 10 nmol 66.9 IMP517 20nmol 85.8 IMP485 20 nmol LSNE Kit 78.3

IMP517 was labeled with F-18 in 400 μL of 1:1 EtOH/saline at differenttemperatures for 15 min.

IMP517 (20 nmol) Isolated % Labeling temp. ° C. Yield 50 5.8 60 19.3 7031.6 90 72.4 100 86.9 110 93.1

FIG. 24 compares the labeling of IMP517 20 nmol kits in 400 μL of 1:1EtOH/saline heated for 15 min. IMP517 gave the highest labeling yieldsof the ligands tested so far and also gave high yields in saline alone.New NODA derivatives with different functional groups in the vicinity ofthe 1,4,7-triazacyclononane ring were prepared and attached to astandard test peptide. The peptides were radiolabeled over a range oftemperatures from 50 to 110° C. with and without a co-solvent. Two ofthese derivatives containing a pyridyl or a triazole group showedimproved labeling yields at lower temperatures as well as labeling equalor better than the benzyl-NODA standard at higher temperatures. Addingethanol to the triazole derivative did not increase yields as much asthe other derivatives, indicating that it may be possible to improve theradiolabeling yield at lower temperatures and reduce or eliminate theneed for a co-solvent.

Alterations to the NODA/NOTA ligand on a peptide can have a positiveeffect on the radiolabeling yield of the peptide, and may lead toligands that can be used for direct one-step ¹⁸F labeling of sometemperature-sensitive molecules.

Example 35 Non-Peptide, Small Molecule-Imaging Agents

A NODA-2-nitroimidazole derivative (50 nmol, I mL) (FIG. 23C) used forhypoxia imaging was labeled in 0.1 M, pH 4, NaOAc buffer by mixing with22.5 μL of 2 mM AlCl₃.6H₂O (45 nmol) in 0.1 M pH 4 NaOAc, and 50 μL of¹⁸F⁻ in saline, then heating at 110° C. for 10 min to obtain the labeledcomplex in 85% yield. In vivo studies with theAl¹⁸F-NODA-2-nitroimidazole showed the expected biodistribution andtumor targeting, with no evidence of product instability. TheNOTA-DUPA-Pep molecule (FIG. 23D) was made for targeting theprostate-specific membrane antigen (PSMA). The ¹⁸F-labeled molecule wassynthesized in 79% yield after HPLC purification to remove the unlabeledtargeting agent.

Example 36 Large Peptide and Protein Labeling

NOTA-N-ethylmaleimide was attached to a cysteine side chain of the 40amino acid exendin-4 peptide, which targets the glucagon-like peptidetype-1 receptor (GLP-1 receptor) (Kiesewetter et al., 2012, Tharanostics2:999-1009). The peptide was labeled with ¹⁸F⁻ using unpurifiedcyclotron target water to obtain the labeled peptide in 23.6±2.4%uncorrected yield in 35 min. The Al¹⁸F-labeled peptide had 15.7±1.4%ID/g in the tumor and 79.25±6.20% ID/g in the kidneys at 30 min, withlow uptake in all other tissues.

The NOTA-affibody Z_(HER2:2395) (58-amino acid, 7 kDa) was labeled at90° C. for 15 min with Al¹⁸F, the affibody, and acetonitrile (Heskamp etal., 2012, J Nucl Med 53:146-53). The labeling and purification processtook about 30 min and the yield was 21±5.7%. Again, biodistributionstudies supported the stability of the product with negligible boneuptake.

We also examined a two-step labeling method for temperature-sensitivemolecules. The NODA-MPAA ligand was attached to N-ethylmaleimide to makeNODA-MPAEM. The NODA-MPAEM (20 nmol in 10 μL 2 mM, pH 4, NaOAc) wasmixed with 5 μL 2 mM AlCl₃ in 2 mM, pH 4, NaOAc followed by 200 μL ¹⁸F⁻in saline and 200 μL of acetonitrile. The solution was heated at105-109° C. for 15 min and purified by SPE to produce theAl¹⁸F-NODA-MPAEM in 80% yield. This product was then coupled to apre-reduced antibody Fab′ fragment (20 nmol) by mixing the purifiedAl¹⁸F-NODA-MPAEM at room temperature for 10 min, followed by isolationof the labeled Fab′ by gel filtration. The labeled protein was obtainedin an 80% yield. The total synthesis time for both steps combined wasabout 50 min, with an overall decay-corrected yield of about 50-60%.

Example 37 Residualization and In Vivo Clearance of Al¹⁸F Complexes

Lang et al. compared the biodistribution of ¹⁸F on carbon, Al¹⁸F and⁶⁸Ga attached to the same NOTA-PRGD2 peptide in the U-87 MG humanglioblastoma model (Lang et al., 2011, Bioconjugate Chem 22:2415-22).They found that tumor uptake of the ¹⁸F-PPRGD2 peptide was 3.65±0.51%ID/g at 30 min PI compared to 1.85±0.30% ID/g at 2 h, indicating thatthe ¹⁸F activity was slowly clearing from the tumor between 30 min and 2h (51% retention). The metal-complexed RGD peptides had higher tumorretention [4.20±0.23% ID/g (30 min), 3.53±0.45% ID/g (2 h) or 84%retention for Al¹⁸F-NOTA-PRGD2, and 3.25±0.62% ID/g (30 min), 2.66±0.32%ID/g (2 h), or 82% retention ⁶⁸Ga-NOTA-PRGD2] over the same period.These data show that the chelated AlF complex may be retained better inthe tumor than the radiofluorinated compound with ¹⁸F bound to a carbonatom. The retention of activity also was seen with the exendin peptideand the affibody, where the activity cleared from the kidneys when the¹⁸F was attached to a carbon atom (Kiesewetter et al., 2012, Eur J NuclMed Mol Imaging 39:463-73; Kramer et al., 2008, Eur J Nucl Med MolImaging 35:1008-18), but was retained with the Al¹⁸F complex(Kiesewetter et al., 2012, Theranostics 2:999-1009; Heskamp et al.,2012, J Nucl Med 53:146-53). Retention of the radionuclide in a tissuecould provide a targeting advantage, particularly in rapidlymetabolizing tissues, such as damaged heart tissue.

What is claimed is:
 1. An ¹⁸F-labeled or ¹⁹F-labeled molecule comprising: a) a complex of ¹⁸F or ¹⁹F and a group IIIA metal; b) a bifunctional chelating agent attached to the ¹⁸F-metal or ¹⁹F-metal complex; and c) a molecule attached to the bifunctional chelating agent to form an ¹⁸F- or ¹⁹F-labeled molecule.
 2. The molecule of claim 1, wherein the bifunctional chelating agent is selected from the group consisting of NODA-BA, NODA-BAEM, NODA-BM, NODA-butyne, NODA-EA, NODA-EBA, NODA-EPA, NODA-EPN, NODA-HA, NODA-MBA, NODA-MBEM, NODA-MPAA, NODA-MPAA NHS ester, NDOA-MPAEM, NODA-MPAPEG₃M₃, NODA-MPH, NODA-MPN, NODA-2-nitroimidazole, NODA-PA, NODA-PAEM and NODA-propyl amine.
 3. The molecule of claim 1, wherein the molecule is selected from the group consisting of a protein, a peptide, an antibody, a monoclonal antibody, a bispecific antibody, a multispecific antibody, an antibody fusion protein, an antigen-binding antibody fragment, an affibody and a targetable construct.
 4. The molecule of claim 1, wherein the molecule is a protein or peptide.
 5. The molecule of claim 3, wherein the molecule binds to an antigen selected from the group consisting of carbonic anhydrase IX, CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CXCR4, CXCR7, CXCL12, HIF-1α, AFP, PSMA, CEACAM5, CEACAM-6, c-met, B7, ED-B of fibronectin, Factor H, FHL-1, Flt-3, folate receptor, GRO-β, HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-1 (ILGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR, tenascin, Le(y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, TNF-α, TRAIL receptor (R1 and R2), VEGFR, EGFR, P1GF, complement factors C3, C3a, C3b, C5a, and C5.
 6. The molecule of claim 1, wherein the molecule is an antibody selected from the group consisting of hR1 (anti-IGF-1R), hPAM4 (anti-pancreatic cancer mucin), hA20 (anti-CD20), hA19 (anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2 (anti-CD22), (anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-CEACAM5), hMN-15 (anti-CEACAM6), hRS7 (anti-EGP-1) and hMN-3 (anti-CEACAM6).
 7. The molecule of claim 1, wherein the molecule is selected from the group consisting of oligonucleotides, hormones, growth factors, cytokines, chemokines, angiogenic factors, anti-angiogenic factors, immunomodulators, peptides, polypeptides, proteins, nucleic acids, antibodies, antibody fragments, interleukins, interferons, oligosaccharides, polysaccharides, lipids, siderophores and vitamins.
 8. The molecule of claim 1, wherein the molecule is somatostatin, EGF, VEGF, bombesin, methotrexate, growth hormone, prostate cancer specific antibody, breast cancer specific antibody, RGD, folic acid, a folic acid derivative or a folic acid analog.
 9. The molecule of claim 1, wherein the molecule is selected from the group consisting of IMP449, IMP460, IMP461, IMP467, IMP469, IMP470, IMP471, IMP479, IMP485, IMP486, IMP487, IMP488, IMP490, IMP493, IMP495, IMP497, IMP500, IMP508 and IMP517.
 10. The molecule of claim 1, wherein the group IIIA metal is selected from the group consisting of aluminum, gallium, indium, and thallium.
 11. The molecule of claim 1, wherein the group IIIA metal is aluminum.
 12. The molecule of claim 11, wherein the aluminum is attached to the bifunctional chelating agent before the ¹⁸F or ¹⁹F is complexed to the aluminum.
 13. The molecule of claim 1, wherein the bifunctional chelating agent is attached to the molecule by a click chemistry reaction or by a maleimide-sulfhydryl reaction.
 14. The molecule of claim 1, wherein the bifunctional chelating agent is conjugated to a molecule via an amide, ester, anhydride, carbonate, carbamate, dithiocarbamate, ether, thioether, disulfide, urea, thiourea, triazoyl, amine, imine, oxime or hydrazone bond.
 15. The molecule of claim 1, wherein a metal-¹⁸F or metal-¹⁹F complex is attached to the bifunctional chelating agent by heating in an aqueous medium at a temperature between 50° C. and 110° C.
 16. The molecule of claim 1, wherein the specific activity of the ¹⁸F-labeled molecule is at least 4,000 Ci/mmol.
 17. The molecule of claim 1, wherein multiple copies of the bifunctional chelating agent are attached to the molecule. 